Methods and compositions useful for inhibition of αvβ5mediated angiogenesis

ABSTRACT

The present invention describes methods for inhibiting angiogenesis in tissues using vitronectin α v β 5  antagonists. The α v β 5 -mediated angiogenesis is correlated with exposure to cytokines including vascular endothelial growth factor, transforming growth factor-α and epidermal growth factor. Inhibition of α v β 5 -mediated angiogenesis is particularly preferred in vascular endothelial ocular neovascular diseases, in tumor growth and in inflammatory conditions, using therapeutic compositions containing α v β 5  antagonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to International ApplicationPCT/US97/09099, filed May 30, 1997, Provisional Application Ser. No.60/018,773, filed May 31, 1996, Provisional Application Ser. No.60/015,869, filed May 31, 1996, which is a CIP of InternationalApplication PCT/US96/13194, filed Aug. 13, 1996, now abandoned, and U.S.application Ser. No. 08/514,799, filed Aug. 14, 1995, now abandoned.

This invention was made with government support under Contract Nos.CA50826, CA45726, HL54444, T32 AIO7244-11 and F32 CA72192 by theNational Institutes of Health. The government has certain rights in theinvention.

TECHNICAL FIELD

The present invention relates generally to the field of medicine, andrelates specifically to methods and compositions for inhibitingα_(v)β₅-mediated angiogenesis of tissues using antagonists of thevitronectin receptor α_(v)β₅.

BACKGROUND

Integrins are a class of cellular receptors known to bind extracellularmatrix proteins, and therefore mediate cell-cell and cell-extracellularmatrix interactions, referred generally to as cell adhesion events.However, although many integrins and their respective ligands aredescribed in the literature, the biological function of many of theintegrins remains elusive. The integrin receptors constitute a family ofproteins with shared structural characteristics of noncovalentheterodimeric glycoprotein complexes formed of α and β subunits.

The vitronectin receptor, named for its original characteristic ofpreferential binding to vitronectin, is now known to refer to threedifferent integrins, designed α_(v)β₁, α_(v)β₃ and α_(v)β₅. Horton, Int.J. Exp. Pathol., 71:741–759 (1990). α_(v)β₁ binds fibronectin andvitronectin. α_(v)β₃ binds a large variety of ligands, including fibrin,fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand'sfactor, osteospontin and bone sialoprotein I. α_(v)β₅ binds vitronectin.The specific cell adhesion roles these three integrins play in the manycellular interactions in tissues are still under investigation. However,it is clear that there are different integrins with different biologicalfunctions as well as different integrins and subunits having sharedbiological specificities.

One important recognition site in a ligand for many integrins is thearginine-glycine-aspartic acid (RGD) tripeptide sequence. RGD is foundin all of the ligands identified above for the vitronectin receptorintegrins. This RGD recognition site can be mimicked by polypeptides(“peptides”) that contain the RGD sequence, and such RGD peptides areknown inhibitors of integrin function. It is important to note, however,that depending upon the sequence and structure of the RGD peptide, thespecificity of the inhibition can be altered to target specificintegrins.

For discussions of the RGD recognition site, see Pierschbacher et al.,Nature, 309:30–33 (1984), and Pierschbacher et al., Proc. Natl. Acad.Sci. USA, 81:5985–5988 (1984). Various RGD polypeptides of varyingintegrin specificity have also been described by Grant et al., Cell,58:933–943 (1989), Cheresh, et al., Cell, 58:945–953 (1989), Aumailleyet al., FEBS Letts., 291:50–54 (1991), and Pfaff et al., J. Biol. Chem.,269:20233–20238 (1994), and in U.S. Pat. Nos. 4,517,686, 4,578,079,4,589,881, 4,614,517, 4,661,111, 4,792,525, 4,683,291, 4,879,237,4,988,621, 5,041,380 and 5,061,693.

Angiogenesis, also referred to as neovascularization, is a process oftissue vascularization that involves the growth of new developing bloodvessels into a tissue. The process is mediated by the infiltration ofendothelial cells and smooth muscle cells. The process is believed toproceed in any one of three ways: 1) The vessels can sprout frompre-existing vessels; 2) De novo development of vessels can arise fromprecursor cells (vasculogenesis); or 3) Existing small vessels canenlarge in diameter. Blood et al., Bioch. Biophys. Acta, 1032:89–118(1990). Vascular endothelial cells are known to contain at least fiveRGD-dependent integrins, including the vitronectin receptor (α_(v)β₃ orα_(v)β₅), the collagen Types I and IV receptor (α₁β₁), the lamininreceptor (α₂β₁), the fibronectin/laminin/collagen receptor (α₃β₁) andthe fibronectin receptor (α₅β₁). Davis et al., J. Cell. Biochem.,51:206–218 (1993). The smooth muscle cell is known to contain at leastsix RGD-dependent integrins, including α₅β₁, α_(v)β₃ and α_(v)β₅.

Angiogenesis is an important process in neonatal growth, but is alsoimportant in wound healing and in the pathogenesis of a large variety ofclinically important diseases including tissue inflammation, arthritis,psoriasis, cancer, diabetic retinopathy, macular degeneration and otherneovascular eye diseases. These clinical entities associated withangiogenesis are referred to as angiogenic diseases. Folkman et al.,Science, 235:442–447 (1987). Angiogenesis is generally absent in adultor mature tissues, although it does occur in wound healing and in thecorpus luteum growth cycle. See, for example, Moses et al., Science,248:1408–1410 (1990).

Inhibition of cell adhesion in vitro using monoclonal antibodiesimmunospecific for various integrin α or β subunits have implicated thevitronectin receptor α_(v)β₃ in cell adhesion of a variety of cell typesincluding microvascular endothelial cells. Davis et al., J. Cell. Biol.,51:206–218 (1993). In addition, Nicosia et al., Am. J. Pathol.,138:829–833 (1991), described the use of the RGD peptide, GRGDS, toinhibit the in vitro formation of “microvessels” from rat aorta culturedin collagen gel.

However, the inhibition of formation of “microvessels” in vitro incollagen gel cultures is not a model for inhibition of angiogenesis in atissue because it is not shown that the microvessel structures are thesame as capillary sprouts or that the formation of the microvessel incollagen gel culture is the same as new-vascular growth into an intacttissue, such as arthritic tissue, tumor tissue or disease tissue whereinhibition of angiogenesis is desirable.

The role of α_(v)β₃ in angiogenesis was recently confirmed. See, Brooks,et al. Science, 264:569–571 (1994). The integrin was shown to beexpressed on blood vessels in human wound granulation tissue but not innormal skin. Monoclonal antibodies against the α_(v)β₁ receptorinhibited angiogenesis induced by the growth factors (cytokines) basicfibroblast growth factor (bFGF) and tumor necrosis factor-α (TNF-α), aswell as by melanoma fragments. However, the antagonists only inhibitednew and not preexisting vessels. In addition, specific linear and cyclicRGD-containing peptides were also shown to inhibit neovascularization.

It has been proposed that inhibition of angiogenesis would be a usefultherapy for restricting tumor growth. Inhibition of angiogenesis hasbeen proposed by (1) inhibition of release of “angiogenic molecules”such as bFGF (basic fibroblast growth factor), (2) neutralization ofangiogenic molecules, such as by use of anti-bFGF antibodies, and (3)inhibition of endothelial cell response to angiogenic stimuli. Thislatter strategy has received attention, and Folkman et al., CancerBiology, 3:89–96 (1992), have described several endothelial cellresponse inhibitors, including collagenase inhibitor, basement membraneturnover inhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D₃ analogs,alpha-interferon, and the like that might be used to inhibitangiogenesis. For additional proposed inhibitors of angiogenesis, seeBlood et al., Bioch. Biophys. Acta., 1032:89–118 (1990), Moses et al.,Science, 248:1408–1410 (1990), Ingber et al., Lab. Invest., 59:44–51(1988), and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744, and5,202,352.

However, the role of the integrin α_(v)β₅ in angiogenesis has neitherbeen suggested or identified until the present invention nor have any ofthe inhibitors of angiogenesis described in the foregoing referencesbeen targeted at inhibition of α_(v)β₅. Moreover, no references, otherthan the present invention, have implicated the α_(v)β₅ integrin inneovascularization, particularly that induced by the growth factors,vascular endothelial growth factor (VEGF), transforming growth factor-α(TGF-α) and epidermal growth factor (EGF).

Although the numbers of growth factors involved in the control ofangiogenesis are limited, different levels of control of the processexist for conversion of a quiescent state to a neovascular state. See,D'Amore, Investigative Ophthal. Visual Sci., 35:3974–3979 (1994). Whilesome growth factors involved in angiogenesis are regulated at thesynthesis level, others are regulated by the state of activation. Thesecellular events occur as a quiescent vessel undergoes neovascularizationfollowing injury or ischemia.

VEGF, in particular, is thought to be a major mediator of angiogenesisin a primary tumor and in ischemic ocular diseases. For review, seeFolkman, Nature Medicine, 1:27–31 (1995). VEGF is a 46 kilodalton (kDa)homodimer that is an endothelial cell-specific angiogenic (Ferrara etal., Endocrin. Rev., 13:18–32 (1992)) and vasopermeable factor (Sengeret al., Cancer Res. 46:5629–5632 (1986)) that binds to high-affinitymembrane-bound receptors with tyrosine kinase activity (Jakeman et al.,J. Clin. Invest., 89:244–253 (1992)).

Activation of receptor tyrosine kinases has recently been shown topromote integrin-dependent cell migration on extracellular matrixproteins. In particular, Klemke et al., J. Cell Biol., 127:859–866(1994) have implicated the EGF receptor (EGFR) tyrosine kinase inpromoting cell motility but not adhesion of FG human pancreaticcarcinoma cells on vitronectin using the α_(v)β₅ integrin. The authorsprovide direct evidence that occupation of EGFR with the EGF ligandactivates the tyrosine kinase activation of the EGFR that ultimatelystimulates a protein kinase C (PKC)-dependent pathway leading to theinduction of α_(v)β₅-dependent cell migration of vitronectin substrateon which the cells are normally unable to migrate. Thus, the Klemke etal. findings provide evidence for correlating the presence of cytokines,specifically EGF, with integrin activity in cell migration. Activationof PKC has been shown to be involved in the regulation of angiogenesisin the chick chorioallantoic membrane model system. See, Tsopanoglou etal., J. Vasc. Res. 30:202–208 (1993). The authors identified specificactivators and inhibitors of PKC that respectively stimulated andinhibited angiogenesis in the model system.

However, neither Klemke et al. nor Tsopanoglou et al. discussed abovedescribe the role of cytokines and expression and/or activation of theα_(v)β₅ integrin in promoting angiogenesis in various conditions anddisease states and inhibition thereof with α_(v)β₅-specific antagonists.

Recent experimental evidence has shown in a monkey model system of eyedisease that retinal ischemia induced by retinal vein occlusion resultedin a rapid rise of VEGF in the aqueous chambers of the eye. This risecoincided with the iris neovascularization that was observed asdescribed by Miller et al., Am. J. Path., 145:574–584 (1994). Additionaldata in an mouse model system of proliferative retinopathy in whichhypoxia is induced, VEGF messenger RNA was shown to increase within 6–12hours of relative hypoxia that remained elevated untilneovascularization developed. As the new blood vessels declined, so didthe VEGF expression as described by Pierce et al., Proc. Natl. Acad.Sci., USA, 92:905–909 (1995).

Thus, the recent data as demonstrated in animal models of ischemia havecorrelated the induction of VEGF with that of ischemia followed byneovascularization. VEGF, as well as other growth factors, have alsobeen implicated in other conditions and disease states involvingneovascularization as reviewed by Folkman, Nature Medicine, 1:27–31(1995).

The Folkman et al. reference also summarizes the current clinicalapproaches used to control undesirable angiogenesis. Patients inclinical trials have received therapeutic treatments with angiogenicinhibitors including platelet factor 4, a fumagillin-derivative,carboxy-aminotriazole, and the like. However, no references or currenttherapeutic references correlate the expression of 60_(v)β₅ withangiogenesis, particularly that induced by VEGF. Thus, prior to thepresent invention, no one has described nor utilized a therapeuticregimen with α_(v)β₅ antagonists to control angiogenesis in a tissueundergoing angiogenesis correlated with the presence and activation ofα_(v)β₅.

Therefore, other than the studies reported here on α_(v)β₃ and therelationship with growth factors to angiogenesis. Applicants are unawareof any other demonstration that angiogenesis could be inhibited in atissue using inhibitors of α_(v)β₅-mediated cell adhesion. Inparticular, it has never been previously demonstrated that α_(v)β₅function is required for angiogenesis in a tissue or that α_(v)β₅antagonists can inhibit angiogenesis in a tissue, particularly in ocularneovascular diseases.

BRIEF DESCRIPTION OF THE INVENTION

The present invention demonstrates that in addition to anα_(v)β₃-requiring angiogenesis pathway in tissues, a separate novelα_(v)β₅-dependent pathway also exists. Thus, the invention describesinhibitors of α_(v)β₅ that can inhibit angiogenesis. The inventionfurther describes that α_(v)β₅-mediated activity in promotingangiogenesis is correlated with growth factor (cytokine) activation ofgrowth factor receptor tyrosine kinases and protein kinase C (PKC). Thegrowth factors (cytokines) that function in this manner include vascularendothelial growth factor (VEGF), transforming growth factor-α (TGF-α),epidermal growth factor (EGF), and the like.

The invention therefore describes methods for inhibiting angiogenesis ina tissue comprising administering to the tissue a composition comprisingan angiogenesis-inhibiting amount of an α_(v)β₅ antagonist.

The tissue to be treated can be any tissue in which inhibition ofangiogenesis is desirable, such as diseased tissue whereneovascularization is occurring. Exemplary tissues include ocular tissueundergoing neovascularization, inflamed tissue, solid tumors,metastases, tissues undergoing restenosis, and the like tissues. Inpreferred embodiments, the neovascularization associated with expressionof α_(v)β₅ is the result of exposure to the growth factors, VEGF, TGF-αand EGF.

Particularly preferred are therapeutic methods directed to inhibitingVEGF-induced vascularization in tissues such as the eye whereangiogenesis is pronounced in diseases, including diabetic retinopathy(also called proliferative diabetic retinopathy), age-related maculardegeneration, presumed ocular histoplasmosis, retinopathy ofprematurity, sickle cell retinopathy and neovascular glaucoma. Infurther preferred embodiments, the therapeutic methods are directed toinhibiting angiogenesis that occurs in corneal neovascular disordersthat include corneal transplantation, herpetic keratitis, luetickeratitis, pterygium, neovascular pannus associated with contact lensuse, and the like.

An α_(v)β₅ antagonist for use in the present methods is capable ofbinding to α_(v)β₅ and competitively inhibiting the ability of α_(v)β₅to bind to the natural vitronectin ligand. Preferably, the antagonistexhibits specificity for α_(v)β₅ over other integrins. In a particularlypreferred embodiment, the α_(v)β₅ antagonist inhibits binding ofvitronectin or other RGD-containing ligands to α_(v)β₅ but does notsubstantially inhibit binding of vitronectin to α_(v)β₃ or α_(IIb)β₃. Apreferred α_(v)β₅ antagonist can be a fusion polypeptide, a linear orcyclic polypeptide, a derivatized polypeptide, a monoclonal antibody ora functional fragment thereof, or an organic molecule that is a mimeticof an α_(v)β₅ ligand that is also referred to as an organic mimetic, allof which specifically interacts with α_(v)β₅.

Administration of the α_(v)β₅ antagonists of this invention includesintraocular, intravenous, transdermal, intrasynovial, intramuscular andoral administration. In other preferred embodiments, administration iscoordinated with a chemotherapeutic regimen to control tumorigenesis andcancer metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIGS. 1A–1D illustrate inhibition of cytokine-induced rabbit cornealangiogenesis by α_(v) integrin antibody antagonists. Induction ofangiogenesis by treatment with either bFGF or VEGF and effects oftreatment thereof with the α_(v) integrin antibody antagonists, P1F6(α_(v)β₅) and LM609 (α_(v)β₃), are described in Example 4. OD and OS arerespectively the right and left eyes of an experimental rabbit. Largearrows indicate corneal angiogenesis with edema while small arrows pointto normal conjunctival limbal vessels. FIGS. 1A and 1B show induction ofangiogenesis with bFGF while FIGS. 1C and 1D show that with VEGF. Rabbitcorneas in FIGS. 1A and 1C show treatment with P1F6 while FIGS. 1B and1D show treatment with LM609.

FIGS. 2A and 2B are histograms showing the mean neovascular area in mm²+/− the standard error (n=8 for each of two series) after inductionrespectively with either bFGF or VEGF followed by mAb treatment witheither P1F6 or LM609. The results are discussed in Example 4.

FIGS. 3A–3F photographically illustrate the effects of anti-integrinantibody treatment on the chick CAM preparation. The results aredescribed in Example 6A. Angiogenesis is either induced with bFGF orVEGF followed by intravenous administration of phosphate buffered saline(PBS) as a control or with P1F6 or LM609 monoclonal antibodies describedin the legend for FIG. 1. CAMs treated with bFGF are shown in FIGS. 3A,3C and 3E while CAMs treated with VEGF are shown in FIGS. 3B, 3D and 3F.Control CAMs receiving intravenous injections of PBS are shown in FIGS.3A and 3B. The P1F6 antibody was used to treat CAMs shown in FIGS. 3Cand 3D while the LM609 antibody was used to treat CAMs in FIGS. 3E and3F.

FIGS. 4A and 4B provide in histogram format the quantitation of resultsshown in FIGS. 3A–3F. The angiogenesis index is plotted on the Y-axisagainst control or antibody treatment. FIGS. 4A and 4B respectively showbFGF- and VEGF-induced angiogenesis. The results are discussed inExample 4.

FIGS. 5A–5F photographically illustrate the effects of synthetic peptidetreatment on the chick CAM preparation as described in Example 6.Angiogenesis is either induced with bFGF or VEGF followed by intravenousadministration of phosphate buffered saline (PBS) as a control or withthe synthetic cyclic peptides RGDfV (SEQ ID NO 4) or RADfV (SEQ ID NO5). CAMs treated with bFGF are shown in FIGS. 5A, 5C and 5E while CAMstreated with VEGF are shown in FIGS. 5B, 5D and 5F. Control CAMsreceiving intravenous injections of PBS are shown in FIGS. 5A and 5B.The RDGfV peptide was used to treat CAMs shown in FIGS. 5C and 5D whilethe RADfV peptide was used to treat CAMs in FIGS. 5E and 5F.

FIGS. 6A and 6B provide, in histogram format, the quantitation ofresults shown in FIGS. 5A–5F. The angiogenesis index is plotted on theY-axis against control or antibody treatment. FIGS. 6A and 6Brespectively show bFGF- and VEGF-induced angiogenesis. The results arediscussed in Example 6.

FIGS. 7A–7E show the effects of anti-integrin monoclonal antibodies andcalphostin C on CAM angiogenesis induced by the separate cytokines,bFGF, TNF-α, VEGF and TGF-α. PMA was also evaluated. The assays andresults are described in Example 6. The results are plotted in histogramformat where angiogenesis index is graphed on the Y-axis and the variouscontrol or inhibitors are shown on the X-axis. FIGS. 7A–7E respectivelyshow angiogenesis induced with bFGF, TNF-α, VEGF, TGF-α and PMA.

FIG. 8 is a histogram showing the effects of antibody treatment on CS1melanoma tumor growth in the chick embryo CAM assayed performed asdescribed in Examples 5C and 6D. The weight of the tumors in milligrams(mg) is plotted on the Y-axis against the various treatments indicatedon X-axis. CSAT is a control antibody specific for the integrin β1subunit. LM609 and P1F6 are previously described.

FIG. 9 is a histogram of the effects of control versus an α_(v)β₅peptide antagonist, labeled peptide 189 (SEQ ID NO 9) on melanoma tumorgrowth as measured by tumor volume in mm³ plotted on the Y-axis. Theassay and results are described in Example 8.

FIG. 10 illustrates the synthesis of Compound 7 as described in Example10A–G.

FIG. 11 illustrates the synthesis of Compound 9 as described in Example10A–C; H–I.

FIG. 12 illustrates the synthesis of Compound 10 as described in Example10J.

FIG. 13 illustrates the synthesis of Compound 12 and Compound 14 asrespectively described in Example 10K–L and 10M–N.

FIG. 14 shows the chemical structures of Compound 15, Compound 16,Compound 17 and Compound 18. The detailed synthesis of said compoundsare described in Example 10O–R.

FIGS. 15A, 15B, 15C and 15D show the consecutive cDNA sequence ofchicken MMP-2 along with the deduced amino acid sequence shown on thesecond line, as shown in FIGS. 15A, 15B and 15C. The third and fourthlines respectively show the deduced amino acid sequence of human andmouse MMP-2 as described in Example 7. The chicken cDNA sequence islisted in SEQ ID NO 23 along with the encoded amino acid sequence thatis also presented separately as SEQ ID NO 24. The numbering of the firstnucleotide of the 5′ untranslated region and region encoding theproenzyme shown in FIG. 15A as a negative number is actually presentedas number 1 in Sequence Listing making the latter appear longer than thefigure; however, the nucleotide sequence is the figure is identical inlength and sequence to that as presented in the listing with theexception of the numbering. Accordingly, references to nucleotideposition for chicken or human MMP-2 in the specification, such as inprimers for use in amplifying MMP-2 fragments, are based on thenucleotide position as indicated in the figure and not as listed in theSequence Listing.

FIG. 16 shows the amino acid residue sequence of mature human MMP-2protein having 631 residues. Amino acid residue positions of humanMMP-2-derived fragments correspond to those in the figure. The aminoacid residue sequence is listed in SEQ ID NO 25.

FIG. 17 shows the effects of peptides 85189 and inert salt counterpart121974 on VEGF-induced angiogenesis in the CAM model as furtherdescribed in Example 6A. The effect is compared to untreated (labeled asNT) and control (labeled as 69601) peptide treated preparations. Theeffect on angiogenesis is measured by calculation of the number ofbranch points as further described in Example 6A.

FIGS. 18, 19 and 20 respectively show the reduction in tumor weight forUCLAP-3, M21-L, and FgM tumors following intravenous exposure to controlpeptide 69601 and antagonist 85189 as further described in Example 6D.The data is plotted with tumor weight on the Y-axis against the peptidetreatments on the X-axis.

FIG. 21 illustrates the effect of peptides and antibodies on melanomatumor growth in the chimeric mouse:human model as further described inExample 8. The peptides assessed included control 69601 (labeled 601)and antagonist 85189 (labeled 189). The antibody tested was LM609. Tumorvolume in mm³ is plotted on the Y-axis against the various treatments onthe X-axis.

FIGS. 22A and 22B respectively show the effect of antagonist 85189(labeled 189) compared to control peptide 69601 (labeled 601) inreducing the volume and wet weight of M21L tumors over a dosage range of10, 50 and 250 μg/injection as further described in Example 8.

FIGS. 23A and 23B show the effectiveness of antagonist peptide 85189(labeled 189 with a solid line and filled circles) against controlpeptide 69601 (labeled 601 on a dotted line and open squares) atinhibiting M21L tumor volume in the mouse:human model with two differenttreatment regimens as further described in Example 8. Tumor volumes inmm³ is plotted on the Y-axis against days on the X-axis.

DETAILED DESCRIPTION OF THE INVENTION A. DEFINITIONS

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552–59 (1969) and adopted at 37 CFR §1.822 (b)(2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other In addition the following have the meanings below:BOC tert-butyloxycarbonyl DCCI dicylcohexylcarbodiimide DMFdimethylformamide OMe methoxy HOBt 1-hydroxybezotriazole

It should be noted that all amino acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino acid residues.

Polypeptide: A linear series of amino acid residues connected to oneanother by peptide bonds between the alpha-amino group and carboxy groupof contiguous amino acid residues.

Peptide: A linear series of no more than about 50 amino acid residuesconnected one to the other as in a polypeptide.

Cyclic peptide: refers to a compound having a heteroatom ring structurethat includes several amide bonds as in a typical peptide. The cyclicpeptide can be a “head to tail” cyclized linear polypeptide in which alinear peptide's n-terminus has formed an amide bond with the terminalcarboxylate of the linear peptide, or it can contain a ring structure inwhich the polymer is homodetic ore heterodetic and comprises amide bondsand/or other bonds to close the ring, such as disulfide bridges,thioesters, thioamides, guanidino, and the like linkages.

Protein: A linear series of greater than 50 amino acid residuesconnected one to the other as in a polypeptide.

Fusion protein: refers to a polypeptide containing at least twodifferent polypeptide domains operatively linked by a typical peptidebond (“fused”), where the two domains correspond to peptides no foundfused in nature.

Synthetic peptide: A chemically produced chain of amino acid residueslinked together by peptide bonds that is free of naturally occurringproteins and fragments thereof.

B. GENERAL CONSIDERATIONS

The present invention relates generally to the discovery thatangiogenesis is mediated by the specific vitronectin receptor α_(v)β₅,and that inhibition of α_(v)β₅ function inhibits angiogenesis. Thisdiscovery is important because of the role that angiogenesis plays in avariety of disease processes. By inhibiting angiogenesis, one canintervene in the disease, ameliorate the symptoms, and in some casescure the disease. Where the growth of new blood vessels is the cause of,or contributes to, the pathology associated with a disease, inhibitionof angiogenesis will reduce the deleterious effects of the disease.Examples include rheumatoid arthritis, diabetic retinopathy,inflammatory diseases, restenosis, and the like. Where the growth of newblood vessels is required to support growth of a deleterious tissue,inhibition of angiogenesis will reduce the blood supply to the tissueand thereby contribute to reduction in tissue mass based on blood supplyrequirements. Examples include growth of new blood vessels in responseto ischemia, resulting in growth factor-induced angiogenesis, growth oftumors where neovascularization is a continual requirement in order thatthe tumor grow beyond a few millimeters in thickness, and for theestablishment of solid tumor metastases.

The methods of the present invention are effective in part because thetherapy is highly selective for angiogenesis and not other biologicalprocesses. As shown in the Examples, only new vessel growth containssubstantial α_(v)β₅, and therefore the therapeutic methods do notadversely effect mature vessels.

The discovery that inhibition of α_(v)β₅ alone will effectively inhibitangiogenesis allows for the development of therapeutic compositions withpotentially high specificity, and therefore relatively low toxicity.Although the invention discloses the use of peptide-based reagents whichhave the ability to inhibit one or more integrins, one can design otherreagents which more selectively inhibit α_(v)β₅. Therefore, certainpeptide-based reagents do not have the side effect of inhibiting otherbiological processes other that those mediated by α_(v)β₅.

For example, as shown by the present teachings, it is possible toprepare monoclonal antibodies highly selective for immunoreaction withα_(v)β₅, and not α_(v)β₁, α_(v)β₃, or α_(IIb)β₃, that are similarlyselective for inhibition of α_(v)β₅ function. In addition,RGD-containing peptides can be designed to be selective for inhibitionof α_(v)β₅, as described further herein.

Prior to the discoveries of the present invention, it was not known thatangiogenesis, and any of the processes dependent on angiogenesis, couldbe inhibited in vivo by the use of reagents that antagonize thebiological function of α_(v)β₅.

C. METHODS FOR INHIBITION OF ANGIOGENESIS

The invention provides for a method of inhibiting angiogenesis in atissue, and thereby inhibiting events in the tissue which depend uponangiogenesis. Generally, the method comprises administering to thetissue a composition comprising an angiogenesis-inhibiting amount of anα_(v)β₅ antagonist.

The target tissue used in practicing the methods of this invention isdefined as α_(v)β₅-containing tissue that is characterized by thedetectable presence of α_(v)β₅ integrin receptor. In other words, anα_(v)β₅-containing tissue is defined by the presence of the α_(v)β₅receptor complex in the cell membranes. Such tissues includeepithelially and mesenchymally derived cells. The presence of thereceptor can be determined by a number of means includingimmunoreactivity of the receptor with an anti-α_(v)β₅ integrin receptorantibody, wherein the immunoreaction is detected in tissues bymicroscopy, by immunoprecipitation, by competition in ligand bindingassays and the like techniques. Preferred antibodies for use indetecting the presence of α_(v)β₅ in a tissue are described below and inExample 1. For example, the distribution of α_(v)β₅ in kidney, skin andocular tissues by immunofluorescence microscopy is described in Example2.

In the context of the methods of this invention, an α_(v)β₅-containingtissue is also characterized as one that has an indicia of angiogenesis.As described earlier, angiogenesis includes a variety of processesinvolving neovascularization of a tissue including “sprouting”,vasculogenesis, or vessel enlargement, all of which angiogenesisprocesses are mediated by and dependent upon the expression of α_(v)β₅.With the exception of traumatic wound healing, corpus luteum formationand embryogenesis, it is believed that the majority of angiogenesisprocesses are associated with disease processes and therefore the use ofthe present therapeutic methods are selective for the disease and do nothave deleterious side effects.

There are a variety of diseases in which angiogenesis is believed to beimportant, referred to as angiogenic diseases, including but not limitedto, inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as restenosis,capillary proliferation in atherosclerotic plaques and osteoporosis, andcancer associated disorders, such as solid tumors, solid tumormetastases, angiofibromas, retrolental fibroplasia, hemangiomas, Kaposisarcoma and the like cancers which require neovascularization to supporttumor growth.

Eye diseases characterized by neovascularization present a particularlypreferred target for therapy. Ocular neovascularization is the mostcommon pathological change observed in the vast majority of eye diseasesthat result in catastrophic loss of vision. The growth of new bloodvessels from the preexisting choroidal, retinal or paralimbal vesselscan lead to edema, hemorrhage or fibrovascular membrane formationresulting in disruption of the normal anatomic relationships of the eyeand concomitant loss of normal visual function.

Eye diseases characterized by angiogenesis include corneal neovasculardisorders that include corneal transplantation, herpetic keratitis,luetic keratitis, pterygium, neovascular pannus associated with contactlens use, and the like. Additional eye diseases also include diabeticretinopathy (DR), age-related macular degeneration (ARMD), presumedocular histoplasmosis (POHS), retinopathy of prematurity (ROP) andneovascular glaucoma and the like. While inhibition of angiogenesis inthese diseases would not necessarily cure the underlying disease, itwould significantly reduce the visual morbidity associated with them.

For example, 90% of the 300,000 persons having diabetes for over 25years will have some form of DR that is a retinal disease characterizedby leaking and/or proliferating blood vessels. Thirty percent of thesepatients will in fact have the latter condition that can be amelioratedwith the therapeutic methods of this invention. For ARDM, 25% of thepopulation over 65, approximately 630,000, will have some form of thedisease with the expectation that by the year 2030, over 6.3 millionindividuals will have ARDM. As a result, having the ability to inhibitα_(v)β₅-associated angiogenesis with the therapeutic compositions andmethods of this invention has great medicinal value.

Thus, methods which inhibit angiogenesis in a diseased tissue amelioratesymptoms of the disease and, depending upon the disease, can contributeto cure of the disease. In one embodiment, the invention contemplatesinhibition of angiogenesis, per se, in a tissue. The extent ofangiogenesis in a tissue, and therefore the extent of inhibitionachieved by the present methods, can be evaluated by a variety ofmethods, such as are described in the Examples for detectingα_(v)β₅-immunopositive nascent and immature vessel structures byimmunohistochemistry.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

In particular, the methods and α_(v)β₅ antagonist compositions of thisinvention are therapeutically useful for inhibiting angiogenesis thathas been induced by growth factors, also referred to as cytokines. Underphysiological conditions, angiogenesis is highly regulated and aspreviously published by Brooks et al., Science, 264:569–5761 (1994), hasbeen shown to be activated by specific angiogenic molecules such asbasic fibroblast growth factor (bFGF). Negative regulators ofangiogenesis have also been described. Angiogenesis is thus regulated byan intricate balance between local stimulators and inhibitors. See,D'Amore, Investigative Ophthal. Visual Sci., 35:3974–3979 (1994).

When the physiologic balance of angiogenic stimulators and inhibitorsthat tightly control the normally quiescent capillary vascular isdisturbed, as occurs is certain disease states, capillary endothelialcells are induced to proliferate, migrate and ultimately differentiateto form new blood vessels.

Angiogenesis is characterized as an event cascade having a set of earlyevents followed by a set of late events as reviewed by Leibovich, “Roleof Cytokines in the Process of Tumor Angiogenesis”, in “Human Cytokines:Their Role in Disease and Therapy”, eds. Aggarwal and Puri, Chapter 35,Blackwell Science, Inc. (1995). The early events are preceded by thedelivery of angiogenic growth factors and cytokines delivered from anextravascular source. The early events then proceed in the targetmicrovasculature with the disruption of intercellular junctions,induction of expression of endothelial cell activation antigens and aproteolytic phenotype, and initiation of endothelial cell migration in adirectional manner. The late events are characterized with autocrine andparacrine expression of growth factor and cytokine genes within thecells, endothelial cells, pericytes and smooth muscle cells, of thedeveloping capillary bud. These cells in turn modulate the interactionsof the cells with the extracellular matrix resulting in the formation ofnew functional capillary loops from existing mature vessels.

As discussed herein and in the Background, reports in the literaturedescribe an association between the appearance of growth factors,including those associated with an increase of α_(v)β₅ expression,namely VEGF, TGF-α and EGF, with the expansion of a tumor mass and inthe onset of angiogenesis in proliferative neovascular eye diseases,both in humans and experimental animals.

Thus, VEGF, EGF, TGF-α, among many others, are considered growth factorswhich are characterized by their properties of stimulating cellulargrowth. Growth factors are proteins that are secreted by one cell thatact on the secreting cell or another cell. Their ability to act isdependent on the presence of growth factor receptors that are usuallytransmembrane proteins. Growth factors such as VEGF are also referred togenerally as cytokines that are defined as polypeptide hormones,secreted by a cell, that affect growth and metabolism either of the same(autocrine) or of another (paracrine) cell. The term cytokine is notlimited to molecules produced by cells of the immune system and thebiological response modifiers of the same system. Thus, the termcytokine is a broad category of which one subcategory based on the typeof biological response is stimulatory growth factors or enhancers suchas VEGF, bFGF, EGF, TGF-α, and the like. For review see, Aggarwal etal., “Common and Uncommon Features of Cytokines and Cytokine Receptors:An Overview”, in “Human Cytokines: Their Role in Disease and Therapy”,eds. Aggarwal and Puri, Chapter 1, Blackwell Science, Inc. (1995).

In the present invention, α_(v)β₅-specific antagonists, and not growthfactor antagonists such as antibodies against VEGF, are contemplated foruse in inhibiting angiogenesis in a tissue. In preferred embodiments,the α_(v)β₅ antagonists described herein are useful for inhibitinggrowth factor-induced angiogenesis in which the expression of theα_(v)β₅ integrin receptor is induced. Preferred growth factors in thiscontext include VEGF, EGF, TGF-α and the like.

As discussed in the Background, the growth factors EGF and VEGF are bothknown to bind to their cellular receptors that act as tyrosine kinases.Activation of the EGF receptor has further been shown to be correlatedwith activation of protein kinase C that results in activation ofα_(v)β₅ to allow for migration of specific cells on a vitronectinsubstrate. Thus, the mechanism of action between exposure to cytokinesor growth factors and the coordinate response in integrin expression oractivation is a complex biological process. As shown in the presentinvention (see Example 6A), treatment of tissues in either the rabbiteye model or the chick chorioallantoic model with the cytokine VEGFresults in the α_(v)β₅-potentiated angiogenesis that is dependent onactivation of protein kinase C.

In a particularly preferred embodiment, the present inventioncontemplates the use of α_(v)β₅ antagonists for inhibiting angiogenesisin any tissue in which angiogenesis has been induced by VEGF. Forexample, ischemia of the retina in various animal model systems has beenshown to result in the upregulation of VEGF that is secreted from Mullercells, the production of which consequently induces neovascularizationof tissues within the eye. See, Miller et al., Am. J. Path., 145:574–584(1994) and Pierce et al., Proc. Natl. Acad. Sci., USA, 92:905–909(1995).

Thus, in the present invention, a tissue to be treated is a retinaltissue of a patient with diabetic retinopathy, macular degeneration,neovascular glaucoma or the like diseases as discussed above and theangiogenesis to be inhibited is retinal tissue angiogenesis where thereis neovascularization of retinal tissue. Exemplary tissues, includingcorneal tissues, from patients with ocular neovascularization conditionsor diseases are described above and in the Examples. An exemplary modelsystem for assessing the effects of an α_(v)β₅ antagonist of thisinvention for treating retinal angiogenesis is the murine model ofretinal neovascularization as described in Example 9.

In another related embodiment, a tissue to be treated is an inflamedtissue and the angiogenesis to be inhibited is inflamed tissueangiogenesis where there is neovascularization of inflamed tissue. Inthis class, the method contemplates inhibition of angiogenesis inarthritic tissues, such as in a patient with chronic articularrheumatism, in immune or non-immune inflamed tissues, in psoriatictissue and the like.

The cytokines, interleukin 1 and tumor necrosis factor-α, are thought tobe associated with rheumatoid arthritis with their direct role in jointdestructions based on the induction of adhesion molecule expression onendothelial cells and on enzyme release. See, Arend et al., Arthritis &Rheumatism, 38:151–160 (1995). Therapeutic regimens have been proposedfor blocking both the cytokines with cytokine-specific inhibitors aswell as targeting cell adhesion molecules that are expressed in thecondition. See, Haskard et al., Cell Adhesion Comm., 2:235–238 (1994).

Thus, inhibition of angiogenesis in arthritic conditions by addressingand directing the therapy to the involvement of the α_(v)β₅ adhesionmolecule is another preferred embodiment of the invention as prior tothis invention.

In an additional related embodiment, a tissue to be treated is a tumortissue of a patient with a solid tumor, a metastases, a skin cancer, abreast cancer, a hemangioma or angiofibroma and the like cancer, and theangiogenesis to be inhibited is tumor tissue angiogenesis where there isneovascularization of a tumor tissue. Typical solid tumor tissuestreatable by the present methods include lung, pancreas, breast, colon,laryngeal, ovarian, and the like tissues.

The role of the complex cytokine network that exists in solid humantumors is the subject of a review by Leek et al., J. Leukocyte Biol.,56:423–435 (1994), the disclosure of which is hereby incorporated byreference. A number of cytokines including VEGF, acidic as well as basicFGF (bFGF), TGF-α and -β, EGF, TNF-α, platelet derived endothelial cellgrowth factor, angiogenin, interferons α and γ, interleukins 1, 6 and 8and the like are thought to influence various cellular mechanisms ofangiogenesis in malignant tissues and cell lines. For example, inaddition to its localization of various kinds of tumors, VEGF hasrecently been shown to be linked to angiogenesis in breast carcinoma asdescribed by Brown et al. Human Path., 26:86–91 (1995).

Tumors that secrete various cytokines and therein induce localizedangiogenesis in response, specifically in the present invention with thecytokines VEGF, TGF-α and EGF and the resultant α_(v)β₅-mediatedangiogenesis, are identifiable by screening tumor tissue samples withanti-cytokine antibodies. Such methods are familiar to one of ordinaryskill in the art for either cultured or biopsied tumor tissue samples.Antibodies against the above-described cytokines are commerciallyavailable through Oncogene Sciences (Uniondale, N.Y.) or Upstate BiotechIncorporated (Lake Placid, N.Y.). The screening of selected tumortissues by these means thereby allows one to assess the potential ofangiogenesis inhibitory activity by the α_(v)β₅ antagonists of thisinvention.

Exemplary tumor tissue angiogenesis, and inhibition thereof, isdescribed in the Examples.

Inhibition of tumor tissue angiogenesis is still another preferredembodiment of the invention because of the important roleneovascularization plays in tumor growth. In the absence ofneovascularization of tumor tissue, the tumor tissue does not obtain therequired nutrients, slows in growth, ceases additional growth, regressesand ultimately becomes necrotic resulting in killing of the tumor.

Stated in other words, the present invention provides for a method ofinhibiting tumor neovascularization by inhibiting tumor angiogenesisaccording to the present methods. Similarly, the invention provides amethod of inhibiting tumor growth by practicing theangiogenesis-inhibiting methods.

The methods are also particularly effective against the formation ofmetastases because (1) their formation requires vascularization of aprimary tumor so that the metastatic cancer cells can exit the primarytumor and (2) their establishment in a secondary site requiresneovascularization to support growth of the metastases. In a relatedembodiment, the invention contemplates the practice of the method inconjunction with other therapies such as conventional chemotherapydirected against solid tumors and for control of establishment ofmetastases. The administration of angiogenesis inhibitor is typicallyconducted during or after chemotherapy, although it is preferable toinhibit angiogenesis after a regimen of chemotherapy at times where thetumor tissue will be responding to the toxic assault by inducingangiogenesis to recover by the provision of a blood supply and nutrientsto the tumor tissue. In addition, it is preferred to administer theangiogenesis inhibition methods after surgery where solid tumors havebeen removed as a prophylaxis against metastases.

Insofar as the present methods apply to inhibition of tumorneovascularization, the methods can also apply to inhibition of tumortissue growth, to inhibition of tumor metastases formation, and toregression of established tumors. For the latter, the diminishment of atumor mass is evaluated in the rabbit eye assay model as described foruse in this invention or with a model system of a chimeric mouse:humanmodel in which skin of a mouse having severe combined immunodeficiency(SCID) is replaced with human neonatal foreskin as described by Yan etal., J. Clin. Invest., 91:986–996 (1993), the disclosure of which ishereby incorporated by reference. The latter model presents anadditional in vivo model to investigate angiogenesis and inhibitionthereof with the methods of this invention. Exemplary results with therabbit tumor model and an α_(v)β₅ antagonists of this invention arepresented in Examples 5C and 6D while results for inbition ofangiogenesis in the SCID mouse model is described in Example 8.

Restenosis is a process of smooth muscle cell (SMC) migration andproliferation at the site of percutaneous transluminal coronaryangioplasty which hampers the success of angioplasty. The migration andproliferation of SMC's during restenosis can be considered a process ofangiogenesis which is inhibited by the present methods. Therefore, theinvention also contemplates inhibition of restenosis by inhibitingangiogenesis according to the present methods in a patient followingangioplasty procedures. For inhibition of restenosis, the α_(v)β₅antagonist is typically administered after the angioplasty procedure forfrom about 2 to about 28 days, and more typically for about the first 14days following the procedure.

The patient treated in the present invention in its many embodiments isdesirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all mammals, which are intended to be included in theterm “patient”. In this context, a mammal is understood to include anymammalian species in which treatment of diseases, particularlyagricultural and domestic mammalian species, is sought with respect tothe methods of this invention.

The present method for inhibiting angiogenesis in a tissue, andtherefore for also practicing the methods for treatment ofangiogenesis-related diseases, comprises contacting a tissue in whichangiogenesis is occurring, or is at risk for occurring, with acomposition comprising a therapeutically effective amount of an α_(v)β₅antagonist capable of inhibiting α_(v)β₅ binding to its natural ligand.Thus, the method comprises administering to a patient a therapeuticallyeffective amount of a physiologically tolerable composition containingan α_(v)β₅ antagonist of the invention.

The dosage ranges for the administration of the α_(v)β₅ antagonistdepend upon the form of the antagonist, and its potency, as describedfurther herein, and are amounts large enough to produce the desiredeffect in which angiogenesis and the disease symptoms mediated byangiogenesis are ameliorated. The dosage should not be so large as tocause adverse side effects, such as hyperviscosity syndromes, pulmonaryedema, congestive heart failure, and the like. Generally, the dosagewill vary with the age, condition, sex and extent of the disease in thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication.

An α_(v)β₅ antagonist is a molecule that blocks or inhibits thephysiologic or pharmacologic activity of α_(v)β₅ by inhibiting thebinding activity of the receptor to its ligand, namely vitronectin.Preferred α_(v)β₅ antagonists can either be a monoclonal antibody, apeptide or an organic-based molecule that is a mimetic of an α_(v)β₅ligand.

A therapeutically effective amount is an amount of α_(v)β₅ antagonistsufficient to produce a measurable inhibition of angiogenesis in thetissue being treated, i.e., an angiogenesis-inhibiting amount.Inhibition of angiogenesis can be measured in situ byimmunohistochemistry, as described herein, or by other methods known toone skilled in the art.

Insofar as an α_(v)β₅ antagonist can take the form of an α_(v)β₅ ligandorganic mimetic, an RGD-containing peptide, an anti-α_(v)β₅ monoclonalantibody, or fragment thereof, or an α_(v)β₅ receptor mimetic it is tobe appreciated that the potency, and therefore an expression of a“therapeutically effective” amount can vary. However, as shown by thepresent assay methods, one skilled in the art can readily assess thepotency of a candidate α_(v)β₅ antagonist of this invention.

Potency of an α_(v)β₅ antagonist can be measured by a variety of meansincluding inhibition of angiogenesis in the CAM assay, in the in vivorabbit eye assay, and by measuring inhibition of binding of naturalligand to α_(v)β₅, all as described herein, and the like assays.

A preferred α_(v)β₅ antagonist has the ability to substantially inhibitbinding of a natural ligand such as vitronectin to α_(v)β₅ in solutionat antagonist concentrations of less than 0.5 micromolar (μM),preferably less than 0.1 μM, and more preferably less than 0.05 μM. By“substantially” is meant that at least a 50 percent reduction in bindingof vitronectin is observed by inhibition in the presence of the α_(v)β₅antagonist, and at 50% inhibition is referred to herein as an IC₅₀value.

A more preferred α_(v)β₅ antagonist exhibits selectivity for α_(v)β₅over other integrins. Thus, a preferred α_(v)β₅ antagonist substantiallyinhibits vitronectin binding to α_(v)β₅ but does not substantiallyinhibit binding of vitronectin to another integrin, such as α_(v)β₁,α_(v)β₃ or α_(IIb)β₃. Particularly preferred is an α_(v)β₅ antagonistthat exhibits a 10-fold to 100-fold lower IC₅₀ activity at inhibitingvitronectin binding to α_(v)β₅ compared to the IC₅₀ activity atinhibiting vitronectin binding to another integrin. Exemplary assays formeasuring IC₅₀ activity at inhibiting vitronectin binding to an integrinare described in the Examples.

A therapeutically effective amount of an α_(v)β₅ antagonist of thisinvention in the form of a monoclonal antibody is typically an amountsuch that when administered in a physiologically tolerable compositionis sufficient to achieve a plasma concentration of from about 0.01microgram (μg) per milliliter (ml) to about 100 μg/ml, preferably fromabout 1 μg/ml to about 5 μg/ml, and usually about 5 μg/ml. Stateddifferently, the dosage can vary from about 0.1 mg/kg to about 300mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, mostpreferably from about 0.5 mg/kg to about 20 mg/kg, in one or more doseadministrations daily, for one or several days.

Where the antagonist is in the form of a fragment of a monoclonalantibody, the amount can readily be adjusted based on the mass of thefragment relative to the mass of the whole antibody. A preferred plasmaconcentration in molarity is from about 2 micromolar (μM) to about 5millimolar (mM) and preferably about 100 μM to 1 mM antibody antagonist.

A therapeutically effective amount of an α_(v)β₅ antagonist of thisinvention in the form of a polypeptide, or other similarly-sized smallmolecule α_(v)β₅ ligand mimetic, is typically an amount of polypeptidesuch that when administered in a physiologically tolerable compositionis sufficient to achieve a plasma concentration of from about 0.1microgram (μg) per milliliter (ml) to about 200 μg/ml, preferably fromabout 1 μg/ml to about 150 μg/ml. Based on a polypeptide having a massof about 500 grams per mole, the preferred plasma concentration inmolarity is from about 2 micromolar (μM) to about 5 millimolar (mM) andpreferably about 100 μM to 1 mM polypeptide antagonist. Stateddifferently, the dosage per body weight can vary from about 0.1 mg/kg toabout 300 mg/kg, and preferably from about 0.2 mg/kg to about 200 mg/kg,in one or more dose administrations daily, for one or several days.

The monoclonal antibodies, polypeptides or organic mimetics of thisinvention can be administered parenterally by injection or by gradualinfusion over time. Although the tissue to be treated can typically beaccessed in the body by systemic administration and therefore most oftentreated by intravenous administration of therapeutic compositions, othertissues and delivery means are contemplated where there is a likelihoodthat the tissue targeted contains the target molecule. Thus, monoclonalantibodies, polypeptides or organic mimetics of this invention can beadministered intraocularly, intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, transdermally, and canalso be delivered by peristaltic means.

The therapeutic compositions containing an α_(v)β₅ antagonist of thisinvention are conventionally administered intravenously, as by injectionof a unit dose, for example. The term “unit dose” when used in referenceto a therapeutic composition of the present invention refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent, i.e., carrier, or vehicle.

In one preferred embodiment as shown in the Examples, the α_(v)β₅antagonist is administered in a single dosage intravenously.

The compositions are administered in a manner compatible with the dosageformulation and in a therapeutically effective amount. The quantity tobe administered and timing of administration depends on the subject tobe treated, capacity of the subject's system to utilize the activeingredient, and degree of therapeutic effect desired. Precise amounts ofactive ingredient required to be administered depend on the judgement ofthe practitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimens for administration arealso variable but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

D. THERAPEUTIC COMPOSITIONS

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with an α_(v)β₅ antagonist as describedherein, dissolved or dispersed therein as an active ingredient. In apreferred embodiment, the therapeutic α_(v)β₅ antagonist composition isnot immunogenic when administered to a mammal or human patient fortherapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Particularly preferred is the HCl salt when used in the preparation ofcyclic polypeptide α_(v)β₅ antagonists.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an angiogenesis-inhibiting amount ofan α_(v)β₅ antagonist of the present invention, typically formulated tocontain an amount of at least 0.1 weight percent of antagonist perweight of total therapeutic composition. A weight percent is a ratio byweight of inhibitor to total composition. Thus, for example, 0.1 weightpercent is 0.1 grams of inhibitor per 100 grams of total composition.

E. ANTAGONISTS OF INTEGRIN α_(v)β₅

α_(v)β₅ antagonists are used in the present methods for inhibitingangiogenesis in tissues, and can take a variety of forms that includecompounds which interact with α_(v)β₅ in a manner such that functionalinteractions with the natural α_(v)β₅ ligands are interfered. Exemplaryantagonists include analogs or mimetics of α_(v)β₅ derived from theligand binding site on α_(v)β₅, mimetics of a natural ligand of α_(v)β₅that mimic the structural region involved in α_(v)β₅-ligand bindinginteractions, polypeptides having a sequence corresponding to afunctional binding domain of the natural ligand specific for α_(v)β₅,particularly corresponding to the RGD-containing domain of a naturalligand of α_(v)β₅, and antibodies which immunoreact with either α_(v)β₅or the natural ligand, all of which exhibit antagonist activity asdefined herein.

1. Polypeptides

In one embodiment, the invention contemplates α_(v)β₅ antagonists in theform of polypeptides. A polypeptide (peptide) α_(v)β₅ antagonist canhave the sequence characteristics of either the natural ligand ofα_(v)β₅ or α_(v)β₅ itself at the region involved in α_(v)β₅-ligandinteraction and exhibits α_(v)β₅ antagonist activity as describedherein. A preferred α_(v)β₅ antagonist peptide contains the RGDtripeptide and corresponds in sequence to the natural ligand in theRGD-containing region.

Preferred RGD-containing polypeptides have a sequence corresponding tothe amino acid residue sequence of the RGD-containing region of anatural ligand of α_(v)β₅ such as vitronectin, for which the sequence iswell known.

A particularly preferred α_(v)β₅ antagonist peptide preferentiallyinhibits α_(v)β₅ binding to its natural ligand(s) when compared to otherintegrins, as described earlier. These α_(v)β₅-specific peptides areparticularly preferred at least because the specificity for α_(v)β₅reduces the incidence of undesirable side effects such as inhibition ofother integrins. The identification of preferred α_(v)β₅ antagonistpeptides having selectivity for α_(v)β₅ can readily be identified in atypical inhibition of binding assay, such as the ELISA assay describedin the Examples.

A polypeptide of the present invention typically comprises no more thanabout 100 amino acid residues, preferably no more than about 60residues, more preferably no more than about 30 residues. Peptides canbe linear or cyclic, although particularly preferred peptides arecyclic. Preferred peptides are described in the Examples.

Where the polypeptide is greater than about 100 residues, it istypically provided in the form of a fusion protein or protein fragment,as described herein.

It should be understood that a subject polypeptide need not be identicalto the amino acid residue sequence of a α_(v)β₅ natural ligand, so longas it includes a sequence necessary for antagonizing the binding of anα_(v)β₅ ligand to α_(v)β₅ and is able to function as an α_(v)β₅antagonist in an assay such as those described herein.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is an α_(v)β₅ antagonist. Therefore, apresent polypeptide can be subject to various changes, substitutions,insertions, and deletions where such changes provide for certainadvantages in its use. In this regard, an α_(v)β₅ antagonist polypeptideof this invention corresponds to, rather than is identical to, thesequence of a recited peptide where one or more changes are made and itretains the ability to function as an α_(v)β₅ antagonist in one or moreof the assays as defined herein.

Thus, a polypeptide can be in any of a variety of forms of peptidederivatives, that includes amides, conjugates with proteins, cyclicpeptides, polymerized peptides, analogs, fragments, chemically modifiedpeptides, and the like derivatives.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the α_(v)β₅ antagonistactivity as described herein. Examples of conservative substitutionsinclude the substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite inhibitionactivity.

A “chemical derivative” refers to a subject polypeptide having one ormore residues chemically derivatized by reaction of a functional sidegroup. In addition to side group derivitations, a chemical derivativecan have one or more backbone modifications including α-aminosubstitutions such as N-methyl, N-ethyl, N-propyl and the like, andα-carbonyl substitutions such as thioester, thioamide, guanidino and thelike. Such derivatized molecules include for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For example: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

A particularly preferred derivative is a cyclic peptide according to theformula cyclo(Arg-Gly-Asp-D-Phe-NMeVal), abbreviated c(RGDf-NMeV), inwhich there is an N-methyl substituted α-amino group on the valineresidue of the peptide and cyclization has joined the primary amino andcarboxy termini of the peptide.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of an α_(v)β₅ natural ligand, it is typicallybecause one or more conservative or non-conservative substitutions havebeen made, usually no more than about 30 number percent, and preferablyno more than 10 number percent of the amino acid residues aresubstituted. Additional residues may also be added at either terminus ofa polypeptide for the purpose of providing a “linker” by which thepolypeptides of this invention can be conveniently affixed to a label orsolid matrix, or carrier.

Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not formα_(v)β₅ ligand epitopes. Typical amino acid residues used for linkingare tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.In addition, a subject polypeptide can differ, unless otherwisespecified, from the natural sequence of an α_(v)β₅ ligand by thesequence being modified by terminal-NH₂ acylation, e.g., acetylation, orthioglycolic acid amidation, by terminal-carboxylamidation, e.g., withammonia, methylamine, and the like terminal modifications. Terminalmodifications are useful, as is well known, to reduce susceptibility byproteinase digestion, and therefore serve to prolong half life of thepolypeptides in solutions, particularly biological fluids whereproteases may be present. In this regard, polypeptide cyclization isalso a useful terminal modification, and is particularly preferred alsobecause of the stable structures formed by cyclization and in view ofthe biological activities observed for such cyclic peptides as describedherein.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid(HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, methane sulfonic acid, acetic acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like. HCl salt is particularly preferred.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

In addition, a peptide of this invention can be prepared as described inthe Examples without including a free ionic salt in which the chargedacid or base groups present in the amino acid residue side groups (e.g.,Arg, Asp, and the like) associate and neutralize each other to form an“inner salt” compound.

A peptide of the present invention also referred to herein as a subjectpolypeptide, can be synthesized by any of the techniques that are knownto those skilled in the polypeptide art, including recombinant DNAtechniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. An excellent summary of the many techniquesavailable can be found in Steward et al., “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al.,“Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976; J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York), 1983; Merrifield, Adv. Enzymol., 32:221–96, 1969;Fields et al., Int. J. Peptide Protein Res., 35:161–214, 1990; and U.S.Pat. No. 4,244,946 for solid phase peptide synthesis, and Schroder etal., “The Peptides”, Vol. 1, Academic Press (New York), 1965 forclassical solution synthesis, each of which is incorporated herein byreference. Appropriate protective groups usable in such synthesis aredescribed in the above texts and in J. F. W. McOmie, “Protective Groupsin Organic Chemistry”, Plenum Press, New York, 1973, which isincorporated herein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently to generate the final linearpolypeptide.

The resultant linear polypeptides prepared, for example, as describedabove may be reacted to form their corresponding cyclic peptides. Anexemplary method for preparing a cyclic peptide is described by Zimmeret al., Peptides 1992, pp. 393–394, ESCOM Science Publishers, B.V.,1993. Typically, tertbutoxycarbonyl protected peptide methyl ester isdissolved in methanol and sodium hydroxide solution are added and theadmixture is reacted at 20° C. (20C) to hydrolytically remove the methylester protecting group. After evaporating the solvent, thetertbutoxycarbonyl protected peptide is extracted with ethyl acetatefrom acidified aqueous solvent. The tertbutoxycarbonyl protecting groupis then removed under mildly acidic conditions in dioxane cosolvent. Theunprotected linear peptide with free amino and carboxy termini soobtained is converted to its corresponding cyclic peptide by reacting adilute solution of the linear peptide, in a mixture of dichloromethaneand dimethylformamide, with dicyclohexylcarbodiimide in the presence of1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclicpeptide is then purified by chromatography.

Alternate methods for cyclic peptide synthesis are described by Gurrathet al., Eur. J. Biochem., 210:911–921 (1992), and described in theExamples.

In addition, the α_(v)β₅ antagonist can be provided in the form of afusion protein. Fusion proteins are proteins produced by recombinant DNAmethods as described herein in which the subject polypeptide isexpressed as a fusion with a second carrier protein such as aglutathione sulfhydryl transferase (GST) or other well known carrier.Preferred fusion proteins comprise an MMP-2 polypeptide describedherein. The preparation of a MMP-2 fusion protein is described in theExamples.

Particularly preferred peptides or derivative peptides for use in thepresent methods in tissues primarily exhibiting α_(v)β₅-associatedangiogenesis are described in the Examples, and include the polypeptidesshown in SEQ ID NOs 4, 6, 7, 8 and 9.

Also preferred are polypeptides derived from MMP-2 described herein,having sequences shown in SEQ ID Nos 11–22.

2. Monoclonal Antibodies

The present invention describes, in one embodiment, α_(v)β₅ antagonistsin the form of monoclonal antibodies which immunoreact with α_(v)β₅ andinhibit α_(v)β₅ binding to its natural ligand as described herein. Theinvention also describes cell lines which produce the antibodies,methods for producing the cell lines, and methods for producing themonoclonal antibodies.

A monoclonal antibody of this invention comprises antibody moleculesthat 1) immunoreact with isolated α_(v)β₅, and 2) inhibit vitronectinbinding to α_(v)β₅. Preferred monoclonal antibodies which preferentiallybind to α_(v)β₅ include a monoclonal antibody having the immunoreactioncharacteristics of mAb P1F6 and mAb P5H9, which are described in theExamples.

The term “antibody or antibody molecule” in the various grammaticalforms is used herein as a collective noun that refers to a population ofimmunoglobulin molecules and/or immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

Exemplary antibodies for use in the present invention are intactimmunoglobulin molecules, substantially intact immunoglobulin moleculesand those portions of an immunoglobulin molecule that contain theparatope, including those portions known in the art as Fab, Fab′,F(ab′)₂ and F(v), and also referred to as antibody fragments.

In another preferred embodiment, the invention contemplates a truncatedimmunoglobulin molecule comprising a Fab fragment derived from amonoclonal antibody of this invention. The Fab fragment, lacking Fcreceptor, is soluble, and affords therapeutic advantages in serum halflife, and diagnostic advantages in modes of using the soluble Fabfragment. The preparation of a soluble Fab fragment is generally knownin the immunological arts and can be accomplished by a variety ofmethods.

For example, Fab and F(ab′)₂ portions (fragments) of antibodies areprepared by the proteolytic reaction of papain and pepsin, respectively,on substantially intact antibodies by methods that are well known. Seefor example, U.S. Pat. No. 4,342,566 to Theofilopolous and Dixon. Fab′antibody portions are also well known and produced from F(ab′)₂ portionsfollowed by reduction of the disulfide bonds linking the two heavy chainportions as with mercaptoethanol, and followed by alkylation of theresulting protein mercaptan with a reagent such as iodoacetamide. Anantibody containing intact immunoglobulin molecules are preferred andare utilized as illustrative herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody bombing sites, each immunospecific for a different epitope,e.g., a bispecific monoclonal antibody.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell carried a hybridoma that secretes (produces)only one kind of antibody molecule. The hybridoma cell is formed byfusing an antibody-producing cell and a myeloma or otherself-perpetuating cell line. The preparation of such antibodies wasfirst described by Kohler and Milstein, Nature, 256:495–497 (1975), thedescription of which is incorporated by reference. Additional methodsare described by Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987). The hybridoma supernates so prepared can then bescreened for the presence of antibody molecules that immunoreact withα_(v)β₅ and for inhibition of α_(v)β₅ binding to natural ligands.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell linesis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a source of α_(v)β₅.

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 G1X⁺ is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of this invention areidentified using the enzyme linked immunosorbent assay (ELISA), avariation of which is described in the Examples.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate specificity. The culture is maintained under conditions andfor a time period sufficient for the hybridoma to secrete the antibodymolecules into the medium. The antibody-containing medium is thencollected. The antibody molecules can then be further isolated by wellknown techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.,8:396, 1959) supplemented with 4.5 gm/l glucose, 20 mM glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of producing a monoclonal antibody, a hybridoma cell or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc. Natl. Acad. Sci., USA, 86:5728–5732(1989) and Huse et al., Science, 245:1275–1281 (1989).

Also contemplated by this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention. Particularly preferred is the hybridoma cell line thatsecretes monoclonal antibody mAb P1F6 and mAb P5H9, the preparation ofwhich is described in the Examples.

The invention contemplates, in one embodiment, a monoclonal antibodythat has the immunoreaction characteristics of mAb P1F6 or mAb P5H9.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same (i.e., equivalent) specificity(immunoreaction characteristics) as a monoclonal antibody of thisinvention by ascertaining whether the former prevents the latter frombinding to a preselected target molecule. If the monoclonal antibodybeing tested competes with the monoclonal antibody of the invention, asshown by a decrease in binding by the monoclonal antibody of theinvention in standard competition assays for binding to the targetmolecule when present in the solid phase, then it is likely that the twomonoclonal antibodies bind to the same, or a closely related, epitope.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the target molecule withwhich it is normally reactive, and then add the monoclonal antibodybeing tested to determine if the monoclonal antibody being tested isinhibited in its ability to bind the target molecule. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

An additional way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to determinethe amino acid residue sequence of the CDR regions of the antibodies inquestion. Antibody molecules having identical, or functionallyequivalent, amino acid residue sequences in their CDR regions have thesame binding specificity. Methods for sequencing polypeptides are wellknown in the art.

The immunospecificity of an antibody, its target molecule bindingcapacity and the attendant affinity the antibody exhibits for theepitope are defined by the epitope with which the antibody immunoreacts.The epitope specificity is defined at least in part by the amino acidresidue sequence of the variable region of the heavy chain of theimmunoglobulin the antibody and in part by the light chain variableregion amino acid residue sequence.

Use of the term “having the binding specificity of” indicates thatequivalent monoclonal antibodies exhibit the same or similarimmunoreaction (binding) characteristics and compete for binding to apreselected target molecule.

Humanized monoclonal antibodies offer particular advantages over murinemonoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens. Inaddition, human antibodies do not activate the immune system in the samemanner as foreign antigens and foreign antibodies. Methods of preparing“humanized” antibodies are generally well known in the art and canreadily be applied to the antibodies of the present invention.

Thus, the invention contemplates, in one embodiment, a monoclonalantibody of this invention that is humanized by grafting to introducecomponents of the human immune system without substantially interferingwith the ability of the antibody to bind antigen.

3. α_(v)β₅-Specific Mimetics

The present invention demonstrates that α_(v)β₅ antagonists generallycan be used in the present invention, the antagonists of which caninclude polypeptides, antibodies and other molecules, designated“mimetics”, that have the capacity to interfere with α_(v)β₅ function.Particularly preferred are antagonists which specifically interfere withα_(v)β₅ function, and do not interfere with function of other integrins.

In this context it is appreciated that a variety of reagents may besuitable for use in the present methods, so long as these reagentspossess the requisite biological activity. These reagents aregenerically referred to a mimetics because they possess the ability to“mimic” an α_(v)β₅ ligand involved in the functional interaction of thereceptor and ligand by blocking the ligand binding domain in thereceptor, and thereby interfere with (i.e., inhibit) normal function. Inan alternative embodiment, an α_(v)β₅ antagonist may be a mimetic of thereceptor rather than its ligand.

A mimetic is any molecule, other than an antibody or ligand-derivedpeptide, which exhibits the above-description properties. It can be asynthetic peptide, an analog or derivative of a peptide, a compoundwhich is shaped like the binding pocket of the above-described bindingdomain such as an organic mimetic molecule, or other molecule.

A preferred mimetic of this invention is an organic-based molecule andthus is referred to as organic mimetic. Particularly preferred organicmimetic molecules that function as α_(v)β₅ antagonists by being amimetic to a ligand of α_(v)β₅ are Compounds 7, 9, 10, 12, 14, 15, 16,17 and 18 as described in Example 10.

The design of an α_(v)β₅ mimetic can be conducted by any of a variety ofstructural analysis methods for drug-design known in the art, includingmolecular modeling, two-dimensional nuclear magnetic resonance (2-D NMR)analysis, x-ray crystallography, random screening of peptide, peptideanalog or other chemical polymer or compound libraries, and the likedrug design methodologies.

In view of the broad structural evidence presented in the presentspecification which shows that an α_(v)β₅ antagonist can be a fusionpolypeptide (e.g., an MMP-2 fusion protein), a small polypeptide, acyclic peptide, a derivative peptide, an organic mimetic molecule, or amonoclonal antibody, that are diversely different chemical structureswhich share the functional property of selective inhibition of α_(v)β₅,the structure of a subject α_(v)β₅ antagonist useful in the presentmethods need not be so limited, but includes any α_(v)β₅ mimetic, asdefined herein.

F. METHODS FOR IDENTIFYING ANTAGONISTS OF α_(v)β₅

The invention also describes assay methods for identifying candidateα_(v)β₅ antagonists for use according to the present methods. In theseassay methods candidate molecules are evaluated for their potency ininhibiting α_(v)β₅ binding to natural ligands, and furthermore areevaluated for their potency in inhibiting angiogenesis in a tissue.

The first assay measures angiogenesis in the chick chorioallantoicmembrane (CAM) and is referred to as the CAM assay. The CAM assay hasbeen described in detail by others, and further has been used to measureboth angiogenesis and neovascularization of tumor tissues. See Ausprunket al., Am. J. Pathol., 79:597–618 (1975) and Ossonski et al., CancerRes., 40:2300–2309 (1980).

The CAM assay is a well recognized assay model for in vivo angiogenesisbecause neovascularization of whole tissue is occurring. Actual chickembryo blood vessels are growing into the CAM or into the tissue grownon the CAM.

As demonstrated herein, the CAM assay illustrates inhibition ofneovascularization based on both the amount and extent of new vesselgrowth. Furthermore, it is easy to monitor the growth of any tissuetransplanted upon the CAM, such as tumor tissue. Finally, the assay isparticularly useful because there is an internal control for toxicity inthe assay system. The chick embryo is exposed to any test reagent. Assuch, the health of the embryo is an indication of toxicity.

The second assay that measures angiogenesis is the in vivo rabbit eyemodel and is referred to as the rabbit eye assay. The rabbit eye assayhas been described in detail by others and further has been used tomeasure both angiogenesis and neovascularization in the presence ofangiogenic inhibitors such as thalidomide. See D'Amato, et al., Proc.Natl. Acad. Sci., USA, 91:4082–4085 (1994).

The rabbit eye assay is a well recognized assay model for in vivoangiogenesis because the neovascularization process, exemplified byrabbit blood vessels growing from the rim of the cornea into the cornea,is easily visualized through the naturally transparent cornea of theeye. Additionally, both the extent and the amount of stimulation orinhibition of neovascularization or regression of neovascularization caneasily be monitored over time.

Finally, the rabbit is exposed to any test reagent and as such thehealth of the rabbit is an indication of toxicity of the test reagent.

The third assay measures inhibition of direct binding of the naturalligand, vitronectin, to α_(v)β₅, and a preferred embodiment is describedin detail in the Examples. The assay typically measures the degree ofinhibition of binding of a natural ligand, such as vitronectin, toisolated α_(v)β₅ in the solid phase by ELISA, the inhibition of which ismediated by an α_(v)β₅-specific inhibition.

Thus, the assay can also be used to identify compounds which exhibitspecificity for α_(v)β₅ and do not inhibit natural ligands from bindingother integrins. The specificity assay is conducted by running parallelELISA assays where both α_(v)β₅ and other integrins are screenedconcurrently in separate assay chambers for their respective abilitiesto bind a natural ligand and for the candidate compound to inhibit therespective abilities of the integrins to bind a preselected ligand.Preferred screening assay formats are described in the Examples.

G. ARTICLE OF MANUFACTURE

The invention also contemplates an article of manufacture which is alabelled container for providing an α_(v)β₅ antagonist of the invention.An article of manufacture comprises packaging material and apharmaceutical agent contained within the packaging material.

The pharmaceutical agent in an article of manufacture is any of theα_(v)β₅ antagonists of the present invention, formulated into apharmaceutically acceptable form as described herein according the thedisclosed indications. The article of manufacture contains an amount ofpharmaceutical agent sufficient for use in treating a conditionindicated herein, either in unit or multiple dosages.

The packaging material comprises a label which indicates the use of thepharmaceutical agent contained therein, e.g., for treating conditionsassisted by the inhibition of angiogenesis, and the like conditionsdisclosed herein. The label can further include instructions for use andrelated information as may be required for marketing. The packagingmaterial can include container(s) for storage of the pharmaceuticalagent.

As used herein, the term packaging material refers to a material such asglass, plastic, paper, foil, and the like capable of holding withinfixed means a pharmaceutical agent. Thus, for example, the packagingmaterial can be plastic or glass vials, laminated envelopes and the likecontainers used to contain a pharmaceutical composition including thepharmaceutical agent.

In preferred embodiments, the packaging material includes a label thatis a tangible expression describing the contents of the article ofmanufacture and the use of the pharmaceutical agent contained therein.

EXAMPLES

The following examples relating to this invention are illustrative andshould not, of course, be construed as specifically limiting theinvention. Moreover, such variations of the invention, now known orlater developed, which would be within the purview of one skilled in theart are to be considered to fall within the scope of the presentinvention hereinafter claimed.

1. Preparation of α_(v)β₅-Specific Monoclonal Antibodies

The monoclonal antibodies, P1F6 and P5H9, were produced using standardhybridoma methods by immunization into RBF/DnJ mice with A549 lungcarcinoma cells as described by Wayner et al., J. Cell Biol.,113:919–929 (1991), the disclosure of which is hereby incorporated byreference. Spleens were removed from the immunized mice and fused withNs-1/FOX-NY myeloma cells. Hybridomas producing antibody directed tocarcinoma cell vitronectin receptors were screened by the specificinhibition of UCLA-P3 adhesion to vitronectin-coated surfaces asdescribed by Wayner et al. and cloned by limiting dilution on thymocytefeeder layers.

Both the P1F6 and P5H9 monoclonal antibodies have been shown tospecifically immunoreact with the α_(v)β₅ complex, and not immunoreactwith α_(v) subunit, with β₅ subunit, or with other integrins. The P1F6monoclonal antibody is commercially available from Gibco BRL (LifeTechnologies, Inc., Gaithersburg, Md.) and the P5H9 monoclonal isavailable from Dr. E. Wayner at the Fred Hutchinson Cancer ResearchInstitute, Seattle, Wash.

Other α_(v)β₅ monoclonal antibodies for use in this invention aresimilarly derived and characterized as described herein. In addition,α_(v)β₅ monoclonal antibodies are produced by fusing spleens isolatedfrom mice that receive immunizations with the α_(v)β₅ receptor in eitheran impure or purified form. Purification of the α_(v)β₅ is a procedurewell known to one or ordinary skill in the art of integrin biology andhas also been described by Smith et al., J. Biol. Chem., 265:11008–11013(1990), the disclosure of which is hereby incorporated by reference.Once purified, the isolated receptor is prepared as an immunogen forimmunizing mice as described in Section E2 and as prepared essentiallyas described by Kohler and Milstein, Nature, 256:495–497 (1975), thedisclosure of which is hereby incorporated by reference. The resultanthybridoma clones are screened for reactivity with the immunogen and arethen characterized as described in the following Examples.

2. Characterization of the Specificity of the Anti-α_(v)β₅ MonoclonalAntibodies and Use in Mapping the Tissue Distribution of α_(v)β₅Expression

A. Specificity for Vitronectin

The P5H9 monoclonal antibody prepared in Example 1 was shown by Wayneret al., J. Cell. Biol., 113:919–929 (1991) to block attachment ofUCLA-P3 carcinoma cells to vitronectin while not affecting cellattachment to collagen or fibronectin. The same cells were also shown tocontain only the α_(v)β₅ vitronectin receptor and not one with α_(v)β₃specificity, immunoprecipitating a heterodimer consisting of an α chain(160 kD) and a β chain (95 kD) with nonreducing conditions. The α_(v)β₅receptor detected by P5H9 was also shown to mediate adhesion of M21melanoma cells and H2981 carcinoma cells to vitronectin. The P1F6monoclonal antibody has the same immunoreactivity profile.

B. Immunofluorescence with Anti-Integrin Receptor Antibodies

During wound healing, the basement membranes of blood vessels expressseveral adhesive proteins, including von Willebrand factor, fibronectin,and fibrin. In addition, several members of the integrin family ofadhesion receptors are expressed on the surface of cultured smoothmuscle and endothelial cells. See, Cheresh, Proc. Natl. Acad. Sci., USA,84:6471 (1987); Janat et al., J. Cell Physiol., 151:588 (1992); andCheng et al., J. Cell Physiol., 139:275 (1989).

In addition to the structure and function of the integrin β₅ subunit,the tissue distribution of the subunit by mapping with other anti-β₅monoclonal antibodies has been described by Pasqualini et al., J. CellSci., 105:101–111 (1993), the disclosure of which is hereby incorporatedby reference.

The β₅ subunit-specific monoclonal antibodies described above, similarto those described in Example 1, were secreted from hybridomas that wereprepared using splenocytes from a mouse that received immunizations withthe A549 human lung carcinoma cell line. The hybridomas were selected bypositive surface staining of A549 cells with the hybridomas culturesupernatant and by immunoprecipitation of α_(v)β₅ complexes fromsurface-labeled A549 extracts. The monoclonal antibodies were then usedto map the tissue distribution of the β₅ subunit in normal human thymus,skin and kidney. Four micron thick sections were cut from the frozentissue blocks on a cryostat microtome for subsequent streptavidin-biotinimmunoperoxidase staining with antibodies specific for the β₅ integrinsperformed as described in the Pasqualini et al. reference.

Staining of thymic sections showed the distribution of β₅ on bloodvessels. Hassal's corpuscles, cortical and medullary stromal cells, andbasement membranes. Skin sections showed β₅ on the basal layer of theepidermis and on some dermal blood vessel walls, and kidney sectionsshowed staining of glomerular regions, juxtaglomerular apparatus,proximal convoluted tubules and collecting tubules. Thus, thedistribution of β₅ is heterogeneous to different cell types includingand, more importantly, on capillary endothelial cells, the staining ofwhich was consistent with staining of cultured umbilical veinendothelial cells.

C. Immunofluorescence of Human Retinal Tissue from Patients with OcularDisease with Anti-Integrin Receptor Antibodies

Ocular neovascularization is the most common pathological changeobserved in the vast majority of eye diseases that result incatastrophic loss of vision. The growth of new blood vessels from thepre-existing choroidal, retinal or paralimbal vessels can lead to edema,hemorrhage or fibrovascular membrane formation resulting in disruptionof the normal anatomic relationships of the eye and concomitant loss ofnormal visual function.

Under physiological conditions, angiogenesis is highly regulated and hasbeen shown to be activated by specific angiogenic cytokines such asbasic fibroblast growth factor (bFGF) and tumor necrosis factor-α(TNF-α). As described by Brooks et al., Science, 264:569–571 (1994),monoclonal antibodies against α_(v)β₃ have been shown to be block bothbFGF- and TNF-α-induced angiogenesis in model systems including the CAMmodel described below. As described in Examples 4–6, monoclonalantibodies against α_(v)β₅ block a separate pathway of angiogenesis,specifically that induced by vascular endothelial growth factor (VEGF),transforming growth factor-α (TGF-α) and epidermal growth factor (EGF).

Thus, as described herein in the context of the present invention, twopathways of angiogenesis are defined by distinct integrins, α_(v)β₃ andα_(v)β₅. To investigate the expression and role of these integrins inhuman ocular disease, epiretinal neovascular membranes and subretinalneovascular membranes were obtained en bloc at vitrectomy from patientswith proliferative diabetic retinopathy (PDR). These patients had beenfollowed clinically and were selected for histological evaluation on thebasis of having active, proliferative neovascular disease documented byclinical examination and fundus fluorescein angiography. The obtainedtissue was frozen immediately in Tissue Tek cryopreservative andsectioned.

When the tissues from these patients were examined byimmunofluorescence, the blood vessels were positive for the integrinα_(v)β₃ as indicated by immunoreactivity with the mouse monoclonalantibody LM609. The distribution of the integrin appeared to berestricted to blood vessels and coincided with staining for a marker ofblood vessels, von Willebrand Factor, as mapped with a rabbit antibodyto the factor. The sites of immunoreactivity were visualized with eitherrhodamine-conjugated anti-mouse immunoglobulin or fluorescein-conjugatedanti-rabbit immunoglobulin, the use of both of which allowedco-localization of the integrin location and blood vessel-specificantibodies.

Specimens obtained from normal eyes or patients with atrophic membranesfree from actively proliferating blood vessels were negative for theintegrin α_(v)β₃ by immunofluorescence.

In parallel, the same tissues were analyzed immunohistochemically forthe presence and distribution of α_(v)β₅ with the anti-α_(v)β₅monoclonal antibody, P1F6, prepared in Example 1. The staining revealedthat α_(v)β₅ was present on blood vessels that co-localized with thedistribution of von Willebrand factor. However, the non-vascular tissuealso displayed limited fluorescence with the P1F6 antibody indicating awider distribution of α_(v)β₅. This was in contrast to the presence ofα_(v)β₃ that was limited to blood vessels.

When immunofluorescent staining of membranes was compared betweenα_(v)β₃ and α_(v)β₅ with the respective antibodies LM609 and P1F6, thepattern of staining on the blood vessel wall was virtually identicalindicating that both α_(v)β₃ and α_(v)β₅ are displayed on the surface ofnewly proliferating human blood vessels present in neovascular eyediseases such as diabetic retinopathy.

The results described herein thus show that the α_(v)β₅ integrinreceptor is selectively expressed in specific tissue types in whichangiogenesis is occurring, such as that seen with neovascular membranesfrom patients having active, proliferative neovascular disease. Thesetissues, along with those tissues exposed to particular growth factorsas described below in Examples 4–6, therefore provided ideal targets fortherapeutic aspects of this invention.

3. Preparation of Synthetic Peptides

a. Synthesis Procedure

The cyclic polypeptides used in practicing the methods of this inventionwere synthesized using standard solid-phase synthesis techniques as, forexample, described by Merrifield, Adv. Enzymol., 32:221–296 (1969), andFields, G. B. and Noble, R. L., Int. J. Peptide Protein Res., 35:161–214(1990).

Two grams (g) of BOC-Arg-Gly-Asp-D-Phe-Val-OMe (SEQ ID NO 1) were firstdissolved in 60 milliliters (ml) of methanol to which was added 1.5 mlof 2 N sodium hydroxide solution to form an admixture. The admixture wasthen stirred for 3 hours at 20 degrees C. (20C). After evaporation, theresidue was taken up in water and acidified to pH 3 with diluted HCl andextracted with ethyl acetate. The extract was dried over Na₂SO₄,evaporated again and the resultant BOC-Arg-Gly-Asp-D-Phe-Val-OH (SEQ IDNO 2) was stirred at 20C for 2 hours with 20 ml of 2 N HCl in dioxane.The resultant admixture was evaporated to obtainH-Arg-Gly-Asp-D-Phe-Val-OH (SEQ ID NO 3) that was subsequently dissolvedin a mixture of 1800 ml of dichloromethane and 200 ml ofdimethylformamide (DMF) followed by cooling to 0C. Thereafter, 0.5 g ofdicyclohexylcarbodiimide (DCCI), 0.3 g of 1-hydroxybenzotriazole (HOBt)and 0.23 ml of N-methylmorpholine were added sequentially with stirring.

The resultant admixture was stirred for another 24 hours at 0C and thenat 20C for yet another 48 hours. The solution was concentrated andtreated with a mixed bed ion exchanger to remove salts. After theresulting resin was removed by filtration, the clarified solution wasevaporated and the residue was purified by chromatography resulting inthe recovery of cyclo(Arg-Gly-Asp-D-Phe-Val) (also listed in singleletter code as c-RGDfV) (SEQ ID NO 4). The lower case letters in thepeptide indicate the D form of the amino acid and not the L form asindicated by capital letters.

The cyclic control peptide, cyclo(Arg-Ala-Asp-D-Phe-Val) (also listed insingle letter code as RADfV) (SEQ ID NO 5) was prepared as describedabove. The cyclic peptide c-RADfV (SEQ ID NO 5) has previously beenshown to inhibiting binding of fibrinogen to the integrin α_(v)β₃, andnot inhibit binding of fibrinogen to the integrins α_(IIb)β₃ or α₅β₁(Pfaff, et al., J. Biol. Chem., 269:20233–20238, 1994).

Other peptides that are specifically inhibitory to the binding ofnatural ligands to α_(v)β₅ are similarly prepared as tested forspecificity and range of activity as described in the followingexamples. These include the following peptides that were analogouslyobtained: cyclo(Gly-D-Arg-Gly-Asp-Phe-Val) (SEQ ID NO 6) andcyclo(Arg-Gly-Asp-Phe-D-Val) (SEQ ID NO 7). The peptides having theamino acid residue sequenceTyr-Thr-Ala-Glu-Cys-Lys-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe (SEQ ID NO8) and cyclo (Arg-Gly-Asp-D-Phe-NMeVal) (SEQ ID NO 9) were alsosynthetically prepared. In SEQ ID NO 9, the prefix “Me” in MeValsignifies that the valine in position 5 is modified by methylation atthe alpha amino nitrogen in the amide bond of the valine residue.

b. Alternate Synthesis Procedure

i. Synthesis of Cyclo-(Arg-Gly-Asp-DPhe-NmeVal), TFA Salt

Fmoc-Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-ONa (SEQ ID NO 41) issynthesized using solid-phase Merrifield-type procedures by sequentiallyadding NMeVal, DPhe, Asp(OBut), Gly and Fmoc-Arg(Mtr) in a step-wisemanner to a 4-hydroxymethyl-phenoxymethyl-polystyrene resin (Wang typeresin) (customary Merrifield-type methods of peptide synthesis areapplied as described in Houben-Weyl, l.c., Volume 15/II, Pages 1 to 806(1974). The polystyrene resin and amino acid residues precursors arecommercially available from Aldrich, Sigma or Fluka chemical companies).After completion of sequential addition of the amino acid residues, theresin is then eliminated from the peptide chain using a 1:1 mixture ofTFA/dichloromethane which provides theFmoc-Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH product (SEQ ID NO 42). TheFmoc group is then removed with a 1:1 mixture of piperidine/DMF whichprovides the crude Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH (SEQ ID NO 43)precursor which is then purified by HPLC in the customary manner.

For cyclization, a solution of 0.6 g ofArg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH (SEQ ID NO 43) (synthesized above)in 15 ml of DMF (dimethylformamide; Aldrich) is diluted with 85 ml ofdichloromethane (Aldrich), and 50 mg of NaHCO₃ are added. After coolingin a dry ice/acetone mixture, 40 μl of diphenylphosphoryl azide(Aldrich) are added. After standing at room temperature for 16 hours,the solution is concentrated. The concentrate is gel-filtered (SephadexG10 column in isopropanol/water 8:2) and then purified by HPLC in thecustomary manner. Treatment with TFA (trifluoroacetic acid)/H₂O (98:2)gives cyclo-(Arg-Gly-Asp-DPhe-NmeVal) (SEQ ID NO 9)×TFA which is thenpurified by HPLC in the customary manner; RT=19.5; FAB-MS (M+H): 589.

ii. Synthesis of “Inner Salt”

TFA salt is removed from the above-produced cyclic peptide by suspendingthe cyclo-(Arg-Gly-Asp-DPhe-NmeVal) (SEQ ID NO 9)×TFA in water followedby evaporation under vacuum to remove the TFA. The cyclic peptide formedis referred to as an “inner salt” and is designatedcyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO 9). The term “inner salt” isused because the cyclic peptide contains two oppositely charged residueswhich intra-electronically counterbalance each other to form an overallnoncharged molecule. One of the charged residues contains an acid moietyand the other charged residue contains an amino moiety. When the acidmoiety and the amino moiety are in close proximity to one another, theacid moiety can be deprotonated by the amino moiety which forms acarboxylate/ammonium salt species with an overall neutral charge.

iii. HCl Treatment to Give Cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO9)×HCl

80 mg of cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO 9) are dissolved in0.01 M HCl five to six times and freeze dried after each dissolvingoperation. Subsequent purification by HPLC givecyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO 9)×HCl; FAB-MS (M+H): 589.

iv. Methane Sulfonic Acid Treatment to GiveCyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO 9)×MeSO₃H

80 mg of cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO 9) are dissolved in0.01 M MeSO₃H (methane sulfonic acid) five to six times and freeze driedafter each dissolving operation. Subsequent purification by HPLC givecyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO 9)×MeSO₃H; RT=17.8; FAB-MS(M+H): 589.

Alternative methods of cyclization include derivatizing the side groupchains of an acyclic peptide precursor with sulfhydryl moieties, andwhen exposed to slightly higher than normal physiological pH conditions(pH 7.5), intramolecularly forms disulfide bonds with other sulfhydrylgroups present in the molecule to form a cyclic peptide. Additionally,the C-terminus carboxylate moiety of an acyclic peptide precursor can bereacted with a free sulfhydryl moiety present within the molecule forproducing thioester cyclized peptides.

TABLE I Peptide Designation Amino Acid Sequence SEQ ID NO 62184(66203*)cyclo(RGDfV) 4 62185(69601*) cyclo(RADfV) 5 62181 cyclo(GrGDFV) 6 62187cyclo(RGDFv) 7 62880 YTAECKPOVTRGDVF 8 121974 (85189*) cyclo (RGDf-NMeV)9 112784 cyclo (RGEf-NMeV) 10 huMMP-2 (410–631)** 11 huMMP-2 (439–631)**12 huMMP-2 (439–512)** 13 huMMP-2 (439–546)** 14 huMMP-2 (510–631)** 15huMMP-2 (543–63l)** 16 chMMP-2 (410–637)*** 17 chMMP-2 (445–637)*** 18chMMP-2 (445–518)*** 19 chMMP-2 (445–552)*** 20 chMMP-2 (516–637)*** 21chMMP-2 (549–637)*** 22 *The peptides designated with an asterisk areprepared in HCl and are identical in sequence to the peptide designatedon the same line; the peptides without an asterisk are prepared in TFA.Lower case letters indicate a D-amino acid; capital letters indicate aL-amino acid.

** The human MMP-2 amino acid residue sequences for synthetic peptidesare indicated by the corresponding residue positions shown in FIGS.15A–15C and also in FIG. 16. (MMP-2 refers to a member of the family ofmatrix metalloproteinase enzymes). The human MMP-2 sequences are listedwith the natural cysteine residues but are not listed with engineeredcysteine residues as described for the fusion peptides. The non-naturalcysteine residues were substituted for the natural amino acid residue atthe indicated residue positions in order to facilitate solubility of thesynthetic as well as expressed fusion proteins and to ensure properfolding for presentation of the binding site.

*** The chicken MMP-2 amino acid residue sequences for syntheticpeptides are indicated by the corresponding residue positions shown inFIGS. 15A–15C. The chicken MMP-2 sequences are listed with the naturalcysteine residues but not with the engineered cysteine residues asdescribed for the fusion peptides as described above.

4. Inhibition of Growth Factor-Induced Angiogenesis with α_(v)β₅Antagonists as Measured by In Vivo Rabbit Eye Model Assay

The effect of anti-α_(v)β₅ antagonists on growth factor-inducedangiogenesis can be observed in naturally transparent structures asexemplified by the cornea of the eye. New blood vessels grow from therim of the cornea, which has a rich blood supply, toward the center ofthe cornea, which normally does not have a blood vessels. Stimulators ofangiogenesis, such as VEGF and TGF-α, when applied to the cornea inducethe growth of new blood vessels from the rim of the cornea. Antagonistsof angiogenesis, applied to the cornea, inhibit the growth of new bloodvessels from the rim of the cornea. Thus, the cornea undergoesangiogenesis through an invasion of endothelial cells from the rim ofthe cornea into the tough collagen-packed corneal tissue which is easilyvisible. The rabbit eye model assay therefore provides in vivo model forthe direct observation of stimulation and inhibition of angiogenesisfollowing the implantation of compounds directly into the cornea of theeye.

A. In Vivo Rabbit Eye Model Assay

1) Angiogenesis Induced by Growth Factors

Angiogenesis was induced in the in vivo rabbit eye model assay withgrowth factors and is described in the following.

a. Preparation of Hydron Pellets Containing Growth Factor and MonoclonalAntibodies

Hydron polymer pellets containing growth factor and monoclonalantibodies (mAbs) were prepared as described by D'Amato, et al., Proc.Natl. Acad. Sci., 91:4082–4085 (1994). The individual pellets contained750 ng of the growth factor (also referred to as cytokine), specificallyeither bFGF or VEGF, bound to sucralfate (carafate) (Carafet, MarionMerrell Down Corporation, Cincinnati, Ohio) to stabilize the cytokinesand ensure their slow release into the surrounding tissue. In addition,hydron pellets were prepared which contained either 40 μg of the mAbP1F6 (anti-α_(v)β₅) or the control antibody, LM609 (anti-α_(v)β₃), inPBS.

All of the mAbs tested were purified from ascites fluid using Protein-ASepharose CL-4B affinity column chromatography according to well-knownmethods. The eluted immunoglobulin was then dialyzed against PBS andtreated with Detoxi-gel (Pierce Chemicals, Rockford, Ill.) to removeendotoxin. Endotoxin has been shown to be potent angiogenic andinflammatory stimulant. Monoclonal antibodies were therefore tested forthe presence of endotoxin with the Chromogenic Limulus Amebocyte LysateAssay (BioWhittaker, Walkersville, Md.) and only those mAbs withoutdetectable endotoxin were used in the rabbit eye model assay.

The pellets were cast in specially prepared Teflon pegs that had a 2.5mm core drilled into their surfaces. Approximately 12 μl of castingmaterial was placed into each peg and polymerized overnight in a sterilehood. Pellets were then sterilized by ultraviolet irradiation.

A series of eight animals were used for paired eye experiments whereeach animal received a Hydron implant containing a preselected cytokinewith a preselected antibody or control immunoglobulin. Specifically, foreach rabbit, one cornea was surgically implanted with a Hydron pelletcontaining either bFGF or VEGF in conjunction with mAb P1F6 and theother cornea was treated with either bFGF or VEGF in conjunction withMAb LM609. Individual pellets were implanted into surgically created“pockets” formed in the mid-stroma of the cornea of rabbits. Thesurgical procedure was done under sterile technique using a Wild modelM691 operating microscope equipped with a beamsplitter to which wasmounted a camera for photographically recording individual corneas. A 3mm by 5 mm “pocket” was created in the corneal stroma by making a 3 mmincision to half the corneal thickness with a 69 Beaver blade. Thestroma was dissected peripherally using an iris spatula and the pelletwas implanted with its peripheral margin 2 mm from the limbus.

During the following 12 days, the cytokines and mAbs diffused from theimplanted pellets into the surrounding tissue thereby effectingangiogenesis from the rim of the cornea.

The left and right corneas are respectively referred to as OS and OD.The corneas were then observed for 12 days. Photographs were taken onpostoperative day 10, the time at which neovascularization is maximal.

Representative photographic results of the above-treatments withcytokine/mAb admixtures are shown in FIGS. 1A–1D. The parallelquantitation of mAb inhibition of cytokine-induced angiogenesis is shownin FIGS. 2A and 2B. In FIGS. 1A and 1D, in which corneas wererespectively exposed to bFGF/P1F6 and VEGF/LM609 combinations,cytokine-induced angiogenesis with edema is prominent as indicated bythe large arrows. Therefore, the α_(v)β₅ antibody, P1F6, was noteffective at inhibiting bFGF-induced angiogenesis. Similarly, theα_(v)β₃ antibody, LM609, was not effective at inhibiting VEGF-inducedangiogenesis.

In contrast, when the cytokine/mAb combinations of bFGF/LM609 andVEGF/P1F6 were used in the rabbit model, the cytokine-inducedangiogenesis was inhibited by the antibodies as shown in FIGS. 1B and1C, respectively. In these figures, normal conjunctival limbal vesselsindicated by the small arrows are shown indicating effectiveness of theintegrin antibodies in inhibiting one type of cytokine-inducedangiogenesis.

The effects of specific mAb integrin immunoreactivity on the abovecytokine-induced angiogenesis is also quantified as shown in FIGS. 2Aand 2B. Angiogenesis was stimulated with either bFGF or VEGF as shownrespectively in FIGS. 2A and 2B. The treated eyes were photographeddaily through a Wild operating microscope outfitted with a Nikon camera.Photographs were recorded on Kodak Ektachrome 64T slide film and imageswere converted for computer-assisted quantitation using Biorad'sMolecular Analyst 1.1 software after acquisition through a Model GS670imaging densitometer. Histograms illustrating the mean neovascular area+/− the standard error (n=8 for each of two series) after exposure tothe mAbs P1F6 or LM609.

As shown in FIG. 2A, LM609 reduced bFGF-induced angiogenesis by 86%(p<0.005, paired t-test) when compared to treatment of the paired eye onthe same animal with P1F6. When VEGF was used to stimulate angiogenesisas shown in FIG. 2B, the opposite effect was observed where P1F6 reducedthe mean area of neovascularization by 60% (p<0.03, paired t-test)compared to the LM609-treated eye that had a minimal effect onVEGF-induced angiogenesis.

Significantly, only the newly cytokine-induced blood vessels wereeffected by exposure to a particular mAb while the pre-existingperilimbal vessels were unaffected by either mAb suggesting that theeffects observed are restricted to newly forming blood vessels of thecornea.

Similar assays are performed with synthetic peptides prepared in Example3 and as described below for use in inhibiting cytokine-inducedangiogenesis that is specifically correlated with α_(v)β₅ expression.

To confirm these results indicating that angiogenesis induced by aparticular cytokine was only effected by one type of anti-integrinantibody, specifically that α_(v)β₅ integrin receptor plays a role inVEGF-induced angiogenesis, another neovascular model of the chickchorioallantoic membrane (CAM) was evaluated with the combinations ofcytokines and integrin antibodies as shown in the next Example.

b. Treatment with Polypeptides

Each experiment consisted of eight rabbits in which one eye received apellet comprising 100 nanograms (ng) bFGF and the other eye received apellet comprising 1 microgram (ug) VEGF. The pellets were inserted intothe corneal pocket as described above, and the cytokines subsequentlystimulated the growth of new blood vessels into the cornea. Peptideswere administered subcutaneously (s.q.) in 1 ml PBS at an initial dosageof 50 ug per kg rabbit the day of pellet insertion, and daily s.q.dosages were given at 20 ug/kg thereafter. After 7 days, the cornea wereevaluated as described above.

Rabbits receiving control peptide 69601 showed substantial corneal bloodvessel growth at 7 days, in both vFGF and VEGF stimulated eyes. Rabbitsreceiving peptide 85189 showed less than 50% of the amount of cornealblood vessel growth compared to controls in vFGF-stimulated eyes andnearly 100% inhibition in VEGF-stimulated eyes.

5. Angiogenesis in the Chick Chorioallantoic Membrane (CAM) Preparation

A. Characterization of the Untreated CAM

1) Preparation of the CAM

Angiogenesis can be induced on the chick chorioallantoic membrane (CAM)after normal embryonic angiogenesis has resulted in the formation ofmature blood vessels. Angiogenesis has been shown to be induced inresponse to specific cytokines or tumor fragments as described byLeibovich et al., Nature, 329:630 (1987) and Ausprunk et al., Am. J.Pathol., 79:597 (1975). CAMs were prepared from chick embryos forsubsequent induction of angiogenesis and inhibition thereof as describedbelow and in Example 6 with the α_(v)β₅ antagonists of this invention.

Ten day old chick embryos were obtained from McIntyre Poultry (Lakeside,Calif.) and incubated at 37C with 60% humidity. A small hole was madethrough the shell at the end of the egg directly over the air sac withthe use of a small crafts drill (Dremel, Division of Emerson ElectricCo., Racine, Wis.). A second hole was drilled on the broad side of theegg in a region devoid of embryonic blood vessels determined previouslyby candling the egg. Negative pressure was applied to the original hole,which resulted in the CAM (chorioallantoic membrane) pulling away fromthe shell membrane and creating a false air sac over the CAM. A 1.0centimeter (cm)×1.0 cm square window was cut through the shell over thedropped CAM with the use of a small model grinding wheel (Dremel). Thesmall window allowed direct access to the underlying CAM.

The resultant CAM preparation was then used at 10 days of embryogenesiswhere angiogenesis has subsided. The preparation was thus used in thisinvention for inducing renewed angiogenesis in response to cytokinetreatment.

2) Histology of the CAM

To analyze the microscopic structure of the chick embryo CAMs, sixmicron (μm) thick sections were cut from the frozen blocks on a cryostatmicrotome for immunofluorescence analysis.

Typical of an untreated 10 day old CAM is an area devoid of bloodvessels. As angiogenesis in the CAM system is subsiding by this stage ofembryogenesis, the system is useful in this invention for stimulatingwith various cytokines the production of new vasculature from existingvessels from adjacent areas into areas of the CAM currently lacking anyvessels.

As shown in the CAM model and in the following Examples, while the bloodvessels are undergoing new growth in normal embryogenesis or induced bycytokines, the blood vessels are expressing α_(v)β₃ and α_(v)β₅.

B. Angiogenesis Induced by Growth Factors

Angiogenesis has been shown to be induced by cytokines or growth factorsas described in Example 4A in the rabbit eye model. In the experimentsdescribed herein, angiogenesis in the rabbit corneal preparationdescribed in Example 4 was similarly induced by growth factors that weretopically applied onto the CAM blood vessels as described herein.

Angiogenesis was induced by placing a 5 millimeter (mm)×5 mm Whatmanfilter disk (Whatman Filter paper No. 1) saturated with Hanks BalancedSalt Solution (HBSS, GIBCO, Grand Island, N.Y.) or HBSS containingpreselected cytokines at a preselected concentration, i.e. one to testthe effect on angiogenesis, on the CAM of a 10 day chick embryo in aregion devoid of blood vessels and the windows were later sealed withtape. Angiogenesis was monitored by photomicroscopy after 72 hours. CAMswere snap frozen then 6 μm cryostat sections were fixed with acetone andstained by immunofluorescence as described in Example 2B and 2C with 10μg/ml of selected anti-integrin antibodies, including those directedagainst α_(v)β₅ as described in Example 1.

Previous studies by Brooks et al., Science, 264:569–571 (1994), haveshown that blood vessels are readily apparent in both the bFGF and TNF-αtreated preparations but are not present in the untreated CAM. Theauthors have also shown that α_(v)β₃ expression was enhanced followingbFGF-induced angiogenesis. While the expression of integrin β₁ did notchange from that seen in an untreated CAM, β₁ was also readilydetectable on stimulated blood vessels.

These published findings indicated that in both human and chick, bloodvessels involved in angiogenesis show enhanced expression of α_(v)β₃.Consistent with this, expression of α_(v)β₃ on cultured endothelialcells were induced by various cytokines in vitro as described by Janatet al., J. Cell Physiol., 151:588 (1992); Enenstein et al., Exp. CellRes., 203:499 (1992) and Swerlick et al., J. Invest. Derm., 99:715(1993).

In this invention, a separate cytokine-mediated pathway for simulatingangiogenesis that is dependent upon expression and activation of adifferent adhesive integrin receptor, α_(v)β₅, has now been determined.The effect of exposure of a CAM as described herein to the cytokinesVEGF, TGF-α and EGF in relationship to the expression of α_(v)β₅, toangiogenesis and inhibition thereof with α_(v)β₅ antagonists isdescribed in Example 6.

C. Angiogenesis Induced by Tumors

To investigate the role of α_(v)β₅ in tumor-induced angiogenesis,various α_(v)β₅-negative human melanoma and carcinoma fragments are usedin the CAM assay that are previously grown and isolated from the CAM of17 day chick embryo as described by Brooks et al., J. Cell Biol.,122:1351 (1993) and as described herein.

Angiogenesis is induced in the CAM assay system by direct apposition ofa tumor fragment on the CAM. Preparation of the chick embryo CAM isidentical to the procedure described above. Instead of a filter paperdisk, a 50 milligram (mg) to 55 mg in weight fragment of oneα_(v)β₅-negative tumor resulting from growth of cell line suspensionsdescribed below, is placed on the CAM in an area originally devoid ofblood vessels.

The cell lines, rabdomyosarcoma, myeloid (HL-60 or KG-1), and lymphoid(T cells—Jurkat, HPB/ALL, PEER, and various B cell lines) as describedby Pasqualini et al. J. Cell Sci., 105:101–111 (1993), are used to growthe solid human tumors on the CAMs of chick embryos. A single cellsuspension of the various cell lines are first applied to the CAMs in atotal volume of 30 μl of sterile HBSS. The windows are sealed with tapeand the embryos are incubated for 7 days to allow growth of human tumorlesions. At the end of 7 days, now a 17 day embryo, the tumors areresected from the CAMs and trimmed free of surrounding CAM tissue. Thetumors are sliced into 50 mg to 55 mg tumor fragments for use inangiogenesis. The tumor fragments are placed on a new set of 10 daychick embryo CAMs as described in Example 5A in an area devoid of bloodvessels.

Tumors grown in vivo on the chick embryo CAMs with and without topicalor intravenous application of α_(v)β₅-inducing cytokines (VEGF, TGF-α,or EGF) are then stained for α_(v)β₅ expression with mAbs, P1F6 or P5H9,as previously described.

These CAM tumor preparations are then subsequently treated as describedin Examples 6C and 6D for measuring the effects of antibodies andpeptides including MMP-2 C-terminal fragments on tumor-inducedangiogenesis.

In one embodiment, hamster melanoma cells, CS-1, obtained from Dr.Caroline Damsky from University of California at San Francisco, wereused in the CAM assay as described above for formation of melanomatumors. Following the transfer of approximately a 50 mg CS-1 tumorfragment on a new 10 day chick embryo CAM, separate preparationsreceived an intravenous injections of either 100 μg or 300 μg of P1F6antibody, LM609 antibody or control CSAT (anti-β1) antibody. Anadditional control included a preparation that received no treatment.The results are discussed below in Example 6D.

6. Inhibition of Angiogenesis as Measured in the CAM Assay

A. Inhibition of Growth Factor-Induced Angiogenesis by IntravenousApplication of Inhibitors

The effect on growth factor-induced angiogenesis with monoclonalantibodies intravenously injected into the CAM preparation was evaluatedfor use as an in vivo model system of this invention.

Following active neovascularization, once the vessels have stoppeddeveloping, the expression of α_(v)β₅ diminishes to levels notdetectable by immunofluorescence analysis. This regulation of α_(v)β₅expression in blood vessels undergoing angiogenesis as contrasted to thelack of expression in mature vessels provides for the unique ability ofthis invention to control and inhibit angiogenesis as shown below asmodeled in the CAM angiogenesis assay system.

The preparation of the chick embryo CAMs for intravenous injections wasessentially as described above.

Angiogenesis was first induced on 10 day old chick embryos byapplication of growth factor-saturated filter disks. Specifically, inthe first assays, angiogenesis was induced by exposure to either bFGF orVEGF, each at a concentration of 150 ng/ml.

For application of growth factors, during the candling procedures,prominent blood vessels were selected and marks were made on the eggshell to indicate their positions. The holes were drilled in the shell,the CAMs were dropped and growth factor-saturated filter papers werethen separately placed on the CAMs as described above. The windows weresealed with sterile tape and the embryos were replaced in the incubator.

Twenty four hours later, a second small window was carefully cut on thelateral side of the egg shell directly over prominent blood vesselsselected previously. The outer egg shell was carefully removed leavingthe embryonic membranes intact. The shell membrane was made transparentwith a small drop of mineral oil (Perkin-Elmer Corp, Norwalk, Conn.)which allowed the blood vessels to be visualized easily. Then, phosphatebuffered saline (PBS), 75 μg of purified sterile anti-integrinantibodies or 75 μg of synthetic peptides (cyclic peptide RGDfV, SEQ IDNO 4 and control cyclic peptide RADfV, SEQ ID NO 5) in PBS were injectedinto blood vessels apparent on the growth factor-induced CAMs. Thewindows were sealed with tape and the embryos were allowed to incubateuntil 72 hours.

The filter discs and representative surrounding CAM tissues werephotographed in a stereomicroscope (FIGS. 3A–3F and FIGS. 5A–5F) and themean angiogenic index +/− the standard error was determined for 12 CAMsper condition (FIGS. 4A–4B and FIGS. 6A–6B). Angiogenesis was scored foreach embryo in a double blind manner by analyzing the number and extentof branching of blood vessels within the area of each disc. The scoresranged from 1 (low) to 4 (high) and the angiogenesis index wasdetermined by subtracting a background of 1 from all data.

Specificity of integrin antibody-mediated inhibition of growthfactor-induced angiogenesis in the CAM model mirrored that seen in therabbit cornea model described above. As respectively shown in FIGS. 3Aand 3B, both bFGF and VEGF caused angiogenesis in the controlPBS-treated CAM. Treatment with the α_(v)β₅-specific antibody, P1F6,however, resulted in inhibition of VEGF-induced angiogenesis as shown inFIG. 3D while no inhibition was detected on bFGF-induced angiogenesis asseen in FIG. 3C. In contrast, the LM609, α_(v)β₃-specific antibodyinhibited bFGF-induced angiogenesis (FIG. 3E) but had little effect onangiogenesis in the VEGF-induced CAM (FIG. 3F).

These results are also shown in the bar graphs of FIGS. 4A and 4B,respectively for both bFGF- and VEGF-treated CAMs, in which theangiogenesis index is plotted against exposure to either LM609 or P1F6along with no antibody exposure as a control. Thus, inhibition of growthfactor-induced angiogenesis by integrin-specific antibodies is dependentupon the type of growth factor.

Exposure to RGD-containing peptides supports the above results. In thepresence of PBS, as shown in FIGS. 5A and 5B, exposure to both bFGF andVEGF resulted in angiogenesis in the control CAM. In contrast, thecyclic peptide antagonist RGDfV (SEQ ID NO 4), directed to both α_(v)β₃and α_(v)β₅, abolished angiogenesis induced by either bFGF or VEGF. Thecyclic peptide RADfV (SEQ ID NO 5) did not effect angiogenesis in eitherthe bFGF- or VEGF-treated CAM preparations. The results are also shownin FIGS. 6A and 6B where the angiogenesis index of bFGF- andVEGF-stimulated CAMS are graphed showing exposure to test and controlpeptides. Thus, these findings together with those in the rabbit corneasindicate that bFGF- and VEGF-induced angiogenesis depend on distinct buthomologous α_(v)-specific integrins that however are both inhibitablewith the cyclic peptide RGDfV.

In a further assay performed in the CAM model having VEGF-inducedangiogenesis, 2 μg of peptides 85189 (SEQ ID NO 9) and the inert saltcounterpart 121974 were separately intravenously injected as previouslydescribed. The effect of the peptides was assessed in comparison to thatof control peptide 69601 (SEQ ID NO 5) and to untreated (labeled as NT)preparations.

The effect of the peptides of VEGF-induced angiogenesis is measuredthrough a determination of the number of blood vessel branch points.Thus, angiogenesis or the lack thereof was quantified by counting thenumber of blood vessels branch points that occur within the confines ofthe filter discs. The branched blood vessels are considered tocorrespond primarily to new angiogenic sprouting blood vessels.Quantification was performed in a double blind manner by at least twoindependent observers. The results are expressed as the Angiogenic Indexwhere the angiogenic index is the number of branch points (VEGFstimulated) minus the number of branch points (control unstimulated) perfilter disc. Experiments routinely had 6–10 embryos per condition. Asshown in FIG. 17, both peptides 85189 and 121974 completely inhibitedangiogenesis as indicated by a reduction of the measurable branch pointsin comparison to untreated or control peptide-treated preparations.

Additional similar assays are performed with synthetic peptides preparedas described in Example 3 to define peptides that exhibit specificity toα_(v)β₅ and not α_(v)β₃ correlated angiogenesis. Assays are alsoperformed with the MMP-2 C-terminal fragments prepared as described inExample 3 and 7 and with the organic molecules prepared as described inExample 10.

The specificity of integrin antibody-inhibition of growth factor-inducedangiogenesis was further confirmed and strengthened by extending thegrowth factor angiogenesis induction analyses to include tumor necrosisfactor-α (TNF-α), transforming growth factor-α (TGF-α) or the phorbolester, 4-β-phorbol-12-myristate-13-acetate (PMA).

The above growth factors (cytokines), including bFGF and VEGF, wereseparately applied at a concentration of 1.0 μg/ml to the 10 day old CAMmodel as previously described. PMA was used at a concentration of 20ng/ml.

After 24 hours after growth factor treatment, the antibodies, LM609 andP1F6, or the protein kinase C (PKC) inhibitor, calphostin C, wereseparately provided to the CAM model, either by a single intravasculardose as described above or by topical administration as described belowin the next example. For intravascular injections over the next 3 dayconsecutive period, the antibodies were used at a concentration of 75 μgper embryo and the calphostin C was at a dosage of 100 nM.

On day 13, filter discs and associated CAM tissue were dissected andanalyzed for angiogenesis with a stereo microscope. Angiogenesis wasscored in a double blind manner by analyzing the number and extent ofbranching of the blood vessels within the area of the discs. The scoresranged from low (1) to high (4). The angiogenesis index was determinedby subtracting a background score of 1 from all data. Experiments wererepeated 2–4 times with 5–6 embryos per condition.

As shown respectively in FIGS. 7A and 7B, the anti-α_(v)β₃ antibody,LM609, blocked angiogenesis in response to bFGF and TNF-α whereas theanti-α_(v)β₅ antibody, P1F6, had little inhibitory effect. In contrast,as shown respectively in FIGS. 7C–7E, P1F6 was effective at inhibitingangiogenesis induced by VEGF, TGF-α, or PMA whereas LM609 failed to doso.

PMA, a potent inducer of angiogenesis, is capable of activating proteinkinase C (PKC), an intracellular family of serine threonine kinases.Therefore, we also examined the effects of calphostin C, a PKCinhibitor, or angiogenesis on the chick CAM. Calphostin C blockedangiogenesis induced by PMA (FIG. 7E) as well as VEGF and TGF-α(respectively shown in FIGS. 7C and 7D) while having minimal effects onbFGF- or TNF-α mediated angiogenesis (respectively shown in FIGS. 7A and7B).

Together, these results indicate the existence of two separate distinctangiogenesis pathways where one is dependent upon an α_(v)β₃-mediatedsignal that is largely independent of PKC, as previously described byBrooks et al., Science, 264:569–571 (1994), and a second pathway ispotentiated by an α_(v)β₅-mediated transduction signal that criticallydepends of PKC activation.

In addition to the above experiments, to determine the localization ofthe P1F6 and LM609 mAbs in CAM tissues that were inoculatedintravenously with LM609, the fixed sections are blocked with 2.5% BSAin HBSS for 1 hour at room temperature followed by staining with a 1:250dilution of goat anti-mouse rhodamine labeled secondary antibody (Tago).The sections are then analyzed with a Zeiss immunofluorescence compoundmicroscope.

B. Inhibition of Growth Factor-Induced Angiogenesis by TopicalApplication of Inhibitors

To determine whether α_(v)β₅ plays an active role in angiogenesis,filter disks saturated with growth factors described above are placed onCAMs to induce angiogenesis followed by application of either P1F6 orLM609.

Disks are then treated with 50 ml HBSS containing 25 mg of mAb in atotal volume of 25 μl of sterile HBSS at 0, 24, and 48 hours. At 72hours, CAMs are harvested and placed in a 35 mm petri dish and washedonce with 1 ml of PBS. The bottom side of the filter paper and CAMtissue is then analyzed under an Olympus stereo microscope, with twoobservers in a double-blind fashion. Angiogenesis inhibition isconsidered significant when CAMs exhibits >50% reduction in blood vesselinfiltration of the CAM directly under the disk. Experiments arerepeated four times per antibody, with 6 to 7 embryos per condition.

To examine the effects of the integrin antibodies on preexisting matureblood vessels present from normal vessel development adjacent to theareas devoid of vessels, filter disks saturated with mAbs are placed onvascularized regions of CAMs from 10 day embryos that do not receivetopical application of cytokine.

CAM assays are also performed with the synthetic peptides of thisinvention to determine the effect of cyclic and linearized peptides ongrowth factor induced angiogenesis. Eight μg of peptides, prepared aspreviously described, are separately presented in a total volume of 25μl of sterile HBSS. The peptide solution is applied to the CAMpreparation immediately and then again at 24 and 48 hrs. At 72 hours thefilter paper and surrounding CAM tissue are dissected and viewed asdescribed above.

Similar assays are performed with the MMP-2 fragments and organicmolecules prepared as respectively described in Examples 7 and 10.

C. Inhibition of Tumor-Induced Angiogenesis by Topical Application

1) Treatment with Monoclonal Antibodies

In addition to the angiogenesis assays described above where the effectsof anti-α_(v)β₅ antibody and peptide antagonists were evaluated, therole of α_(v)β₅ in tumor-induced angiogenesis is also investigated. Asan inducer, α_(v)β₅-negative human tissues previously grown and isolatedfrom the CAM of a 17-day chick embryo are used. The fragments areprepared as described in Example 5C.

As described above, mAbs are separately topically applied to the tumorfragments at a concentration of 25 μg in 25 μl of HBSS and the windowsare then sealed with tape. The mAbs are added again in the same fashionat 24 hours and 48 hours. At 72 hours, the tumors and surrounding CAMtissues are analyzed as described above.

As described in Example 5C, tumors are initially derived bytransplanting human cell lines, which do not express integrin α_(v)β₅,onto the CAMs of 10 day old chick embryos.

In order to quantitate the effect of the mAbs on the tumor-inducedangiogenesis, blood vessels entering the tumor within the focal plane ofthe CAM are counted under a stereo microscope by two observers in adouble-blind fashion.

The synthetic peptides prepared in Example 3, the MMP-2 preparationsdescribed in Example 7 and the organic molecules prepared in Example 10are similarly topically applied to the tumor-induced angiogenic CAMassay system as described above. The effect of the peptides includingthe MMP-2 preparations and organic molecules described in this inventionon the viability of the vessels is similarly assessed.

D. Inhibition of Tumor-Induced Angiogenesis by Intravenous Application

1) Treatment with Monoclonal Antibodies

Tumor-induced blood vessels prepared above were also treated with mAbsapplied by intravenous injection. CS-1 melanoma tumors were placed onthe CAMs as described in Example 5C and the windows were sealed withtape and 24 hours later, 100 to 300 μg of purified mAbs were inoculatedonce intravenously in chick embryo blood vessels as describedpreviously. The chick embryos were then allowed to incubate for 7 days.The extent of angiogenesis was then observed as described in above.After this time period, the tumors were resected and analyzed by theirweight to determine the effect of antibody exposure on tumor growth orsuppression.

The results of treatment of CS-1 tumors with 300 μg of α_(v)β₅ specificantibody P1F6 are shown in FIG. 8. The tumor weight was dramaticallyreduced to less than 50 mg as compared to untreated to CSAT-treatedtumors. The α_(v)β₅ specific antibody, LM609, also inhibited tumorgrowth, however, less effective than that with P1F6. Comparable resultswere obtained with tumors receiving treatment with 100 μg of P1F6. Thus,P1F6 was effective at inhibiting α_(v)β₅-mediated angiogenesis in atumor model on a CAM preparation resulting in a diminution of tumor cellmass.

2) Treatment with Other α_(v)β₅ Antagonists

The effects of peptides, MMP-2 preparations or organic molecules ontumor-induced vasculature in the CAM assay system are also assessed. Thetumor-CAM preparation is used as described above with the exception thatinstead of intravenous injection of a mAb, synthetic peptides includingMMP-2 preparations prepared as described in Example 7 and organicmolecules prepared in Example 10 are separately intravenously injectedinto visible blood vessels.

In one particular set of assays, additional tumor regression assays wereperformed with the α_(v)β₅-reactive peptide 85189 (SEQ ID NO 9) against69601 (SEQ ID NO 5) as a control. The assays were performed as describedabove with the exception that 100 ug of peptide was intravenouslyinjected into the CAM at 18 hourst postimplantation of the varioustumors which in this case included UCLAP-3, M21-L and FgM tumor types.After 48 hours more, the tumors were then resected and wet weights wereobtained.

FIGS. 18, 19 and 20 respectively show the reduction in tumor weight forUCLAP-3, M21-L and FgM tumors following intravenous exposure to peptide85189 in contrast to the lack of effect with either PBS or peptide69601.

7. Identification of α_(v)β₅-Specific Antagonists Detected by Inhibitionof Cell Attachment and by a Ligand-Receptor Binding Assay

A. Inhibition of Cell Attachment

As one means to determine integrin receptor specificity of theantagonists of this invention, inhibition of cell attachment assays areperformed as described below.

Briefly, CS-1 hamster melanoma cells lacking expression of α_(v)β₃ andα_(v)β₅ are first transfected with an plasmid for expressing the β₅subunit as previously described by Filardo et al., J. Cell Biol.,130:441–450 (1995). Specificity of potential α_(v)β₅ antagonists wasdetermined by the ability to block the binding of α_(v)β₅-expressingCS-1 cells to VN or laminin coated plates. As an example of a typicalassay, the wells were first coated with 10 ug/ml substrate overnight.After rinsing and blocking with 1% heat-denatured BSA in PBS at roomtemperature for 30 minutes, peptide 85189 (SEQ ID NO 9) over aconcentration range of 0.0001 uM to 100 uM, are separately mixed withCS-1 cells for applying to wells with a cell number of 50,000cells/well. After a 10–15 minute incubation at 37C, the solutioncontaining the cells and peptides is discarded. The number of attachedcells is then determined following staining with 1% crystal violet. Cellassociated crystal violet is eluted by the addition of 100 microliters(μl) of 10% acetic acid. Cell adhesion was quantified by measuring theoptical density of the eluted crystal violet at a wave length of 600 nm.

Similar assays are performed with fusion proteins or the syntheticpeptide counterparts containing various regions of the MMP-2 protein.The MMP-2-derived polypeptides include regions of the C-terminus ofMMP-2 active in the binding interaction with α_(v)β₅ and thereby capableof inhibiting MMP-2 activation and associated activities. Thesepolypeptides are prepared either as synthetic polypeptides having asequence derived from the C-terminal domain of MMP-2 as described inExample 1 or as fusion proteins including all or a portion of theC-terminal domain of MMP-2, prepared as described below. MMP-2C-terminal molecules are presented for both chicken and human specificsequences.

The chicken-derived MMP-2 C-terminal domain, also referred to as thehemopexin domain immediately contiguous with the hinge region, comprisesthe amino acid residues 445–637 of MMP-2. The complete nucleotide andencoded amino acid sequence of chicken MMP-2 is described below and isshown in FIGS. 15A–15D, with the nucleotide and amino acid sequencesrespectively listed as SEQ ID NOs 23 and 24. The human MMP-2 nucleotideand encoded amino acid sequence is also described below, with the lattershown in FIG. 16 and SEQ ID NO 25. The C-terminal domain in the humanMMP-2 that corresponds to the chicken region of 445–637 begin at aminoacid residue 439 and ends with 631 due to six missing residues from thehuman sequence as shown in FIG. 15C. Both human- and chicken-derivedC-terminal MMP-2 synthetic peptides for use in practicing the methods ofthis invention are listed in Table 1. The amino acid residue sequencesof the synthetic peptides are the same as those generated by therecombinant fusion protein counterparts but without the GST fusioncomponent. The C-terminal MMP-2 fusion proteins derived from bothchicken and human are prepared as described below.

A MMP-2 fusion protein is a chimeric polypeptide having a sequence ofMMP-2 C-terminal domain or a portion thereof fused (operatively linkedby covalent peptide bond) to a carrier (fusion) protein, such asglutathione sulfhydryl transferase (GST).

To amplify various regions of chicken and human MMP-2, primer sequenceswere designed based on the known respective cDNA sequences of chickenand human MMP-2. The complete top strand of the cDNA nucleotide sequenceof unprocessed chicken MMP-2, also referred to as progelatinase, isshown in FIGS. 15A–15D along with the deduced amino acid sequence shownon the second line (Aimes et al., Biochem. J., 300:729–736, 1994). Thethird and fourth lines of the figure respectively show the deduced aminoacid sequence of human (Collier et al., J. Biol. Chem., 263:6579–6587(1988)) and mouse MMP-2 (Reponen et al., J. Biol. Chem., 267:7856–7862(1992)). Identical residues are indicated by dots while the differingresidues are given by their one letter IUPAC lettering. Missing residuesare indicated by a dash. The numbering of the amino acid residues startsfrom the first residue of the proenzyme, with the residues of the signalpeptide being given negative numbers. The nucleotide sequence isnumbered accordingly in the figure although in the Sequence Listing, thefirst nucleotide is listed as number 1. The putative initiation oftranslation (ATG) is marked with three forward arrowheads and thetranslation termination signal (TGA) is indicated by an asterisk. Theamino terminal sequences for the chicken proenzyme and active enzyme arecontained with diamonds and single arrowheads. As previously stated, thechicken progelatinase nucleotide and amino acid residue sequences arelisted together as SEQ ID NO 23 while the encoded amino acid residuesequence is listed separately as SEQ ID NO 24.

Templates for generating amplified regions of chicken MMP-2 were eithera cDNA encoding the full-length mature chicken MMP-2 polypeptideprovided by Dr. J. P. Quigley of the State University of New York atStoney Brook, N.Y. or a cDNA generated from a total cellular RNAtemplate derived by standard techniques from an excised sample ofchicken chorioallantoic membrane tissue. For the latter, the cDNA wasobtained with MuLV reverse transcriptase and a downstream primerspecific for the 3′-terminal nucleotides. 5′ATTGAATTCTTCTACAGTTCA3′ (SEQID NO 26), the 5′ and 3′ ends of which was respectively complementary tonucleotides 1932–1912 of the published chick MMP-2 sequence. Asdescribed in the FIG. 15 figure legend, the nucleotide positions of theprimers described herein correspond to those shown in the figure and notas shown in the Sequence Listing as the latter begins with number 1 andnot as a negative number as indicated in the figure. Reversetranscriptase polymerase chain reaction (RT-PCR) was performed accordingto the specifications of the manufacturer for the GeneAmp RNA PCR Kit(Perkin Elmer). The primer was also engineered to contain an internalEcoRI restriction site.

From either of the above-described cDNA templates, a number ofC-terminal regions of chicken MMP-2, each having the natural cysteineresidue at position 637 at the carboxy terminus, were obtained by PCRwith the 3′ primer listed above (SEQ ID NO 26) paired with one of anumber of 5′ primers listed below. The amplified regions encoded thefollowing MMP-2 fusion proteins, having sequences corresponding to theamino acid residue positions as shown in FIGS. 15B and 15C and alsolisted in SEQ ID NO 24: 1) 203–637; 2) 274–637; 3) 292–637; 4) 410–637;5) 445–637. Upstream or 5′ primers for amplifying each of the nucleotideregions for encoding the above-listed MMP-2 fusion proteins weredesigned to encode the polypeptide start sites 3′ to an engineered,i.e., PCR-introduced, internal BamHI restriction site to allow fordirectional ligation into either pGEX-1λT or pGEX-3X expression vectors.The 5′ primers included the following sequences, the 5′ and 3′ ends ofwhich correspond to the indicated 5′ and 3′ nucleotide positions of thechicken MMP-2 sequence shown in the figure (the amino acid residueposition start sites are also indicated for each primer): 1) Nucleotides599–619, encoding a 203 start site 5′ATGGGATCCACTGCAAATTTC3′ (SEQ ID NO27); 2) Nucleotides 809–830, encoding a 274 start site5′GCCGGATCCATGACCAGTGTA3′ (SEQ ID NO 28); 3) Nucleotides 863–883,encoding a 292 start site 5′GTGGGATCCCTGAAGACTATG3′ (SEQ ID NO 29); 4)Nucleotides 1217–1237, encoding a 410 start 5′AGGGGATCCTTAAGGGGATTC3′(SEQ ID NO 30); and 5) Nucleotides 1325–1345, encoding a 445 start site5′CTCGGATCCTCTGCAAGCACG3′ (SEQ ID NO 31).

The indicated nucleotide regions of the template cDNA were subsequentlyamplified for 35 cycles (annealing temperature 55C) according to themanufacturer's instructions for the Expand High Fidelity PCR System(Boehringer Mannheim). The resulting PCR products were gel-purified,digested with BamHI and EcoRI restriction enzymes, and repurified beforeligation into either pGEX-1λT or pGEX-3X vector (Pharmacia Biotech,Uppsala, Sweden) which had been similarly digested as well asdephosphorylated prior to the ligation reaction. The choice of plasmidwas based upon the required reading frame of the amplification product.Competent E. coli strain BSJ72 or BL21 cells were transformed with theseparate constructs by heat shock. The resulting colonies were screenedfor incorporation of the respective MMP-2 fusion protein-encodingplasmid by PCR prior to dideoxy sequencing of positive clones to verifythe integrity of the introduced coding sequence. In addition,verification of incorporation of plasmid was confirmed by expression ofthe appropriately-sized GST-MMP-2 fusion protein.

Purification of each of the recombinant GST-MMP-2 fusion proteins wasperformed using IPTG-induced log-phase cultures essentially as describedby the manufacturer for the GST Gene Fusion System (Pharmacia Biotech).Briefly, recovered bacteria were lysed by sonication and incubated withdetergent prior to clarification and immobilization of the recombinantprotein on sepharose 4B-coupled glutathione (Pharmacia Biotech). Afterextensive washing, the immobilized fusion proteins were separatelyeluted from the affinity matrix with 10 mM reduced glutathione in 50 mMTris-HCl, pH 8.0, and dialyzed extensively against PBS to removeresidual glutathione prior to use.

Prior attempts to produce fusion proteins between chicken MMP-2 residues445 and 637 that only had one encoded cysteine residue resulted ininsoluble products. Therefore, in order to generate additional solubleMMP-2 fusion proteins derived from the C-terminal region that did notinclude an endogenous terminal cysteine residue as present in thepreviously-described fusion protein, nucleotide sequences wereintroduced into amplified MMP-2 regions to encode a cysteine residue ifnecessary depending on the particular fusion protein. A cysteine residueis naturally present in the chicken MMP-2 sequence at position 446 andat position 637. In the human sequence, these positions correspondrespectively to 440 and 631. Therefore, fusion proteins were designed tocontain engineered terminal cysteine residues at the amino- orcarboxy-terminus of the chicken MMP-2 sequences of interest so as toprovide for disulfide-bonding with the naturally occurring cysteine atthe other terminus, as required by the construct. Synthetic MMP-2fragments for both chicken and human are similarly prepared aspreviously described in Example 3.

Oligonucleotide primers were accordingly designed to allow foramplification of chicken MMP-2 C-terminal regions for expression ofsoluble MMP-2/GST fusion proteins. Amplified chicken MMP-2 C-terminalregions included those for encoding amino acid residue positions445–518, 445–552, 516–637 and 549–637. For fusion proteins containingresidue 517, the naturally encoded tyrosine residue was substituted fora cysteine to allow for disulfide bonding with either cysteine atresidue position 446 or 637. For fusion proteins containing residue 551,the naturally encoded tryptophan residue was substituted for a cysteineto allow for disulfide bonding with either naturally encoded cysteine atresidue position 446 or 637.

Briefly, the pGEX-3X plasmid construct encoding the recombinantGST/MMP-2(410–637) fusion protein prepared above was used as a templatefor amplification according to the manufacturer's protocol for theExpand High Fidelity PCR Kit (Boehringer Mannheim) utilizing a set ofoligonucleotide primers whose design was based on the published chickenMMP-2 sequence (also shown in FIGS. 15A–15D and in SEQ ID NO 23). Oneupstream primer, designed to encode a chicken MMP-2 protein start siteat position 445 after an engineered internal BamHI endonucleaserestriction site for insertion into the pGEX-3X GST vector, had thenucleotide sequence (5′CTCGGATCCTCTGCAAGCACG3′ (SEQ ID NO 32)). The 5′and 3′ ends of the primer respectively corresponded to positions1325–1345 of the chicken MMP-2 sequence in FIG. 15C. Another upstreamprimer, designed to encode a chicken MMP-2 protein start site atposition 516 after an engineered internal BamHI restriction site forinsertion into the pGEX-1λT GST vector and to encode a cysteine residueat position 517, had the nucleotide sequence(5′GCAGGATCCGAGTGCTGGGTTTATAC3′ (SEQ ID NO 33)). The 5′ and 3′ ends ofthe primer respectively corresponded to positions 1537–1562 of thechicken MMP-2 sequence in the figure. A third upstream primer, designedto encode a chicken MMP-2 protein start site at position 549 followingan engineered internal EcoRI endonuclease restriction site for insertioninto the pGEX-1λT GST vector and to encode a cysteine residue atposition 551, had the nucleotide sequence(5′GCAGAATTCAACTGTGGCAGAAACAAG3′ (SEQ ID NO 34)). The 5′ and 3′ ends ofthe primer respectively corresponded to positions 1639–1665 of thechicken MMP-2 sequence in the figure.

These upstream primers were separately used with one of the followingdownstream primers listed below to produce the above-described regionsfrom the C-terminal domain of chicken MMP-2. A first downstream primer(antisense), designed to encode a chicken MMP-2 protein termination siteat position 518, to encode a cysteine residue at position 517, and tocontain an internal EcoRI endonuclease restriction site for insertioninto a GST vector, had the nucleotide sequence(5′GTAGAATTCCAGCACTCATTTCCTGC3′ (SEQ ID NO 35)). The 5′ and 3′ ends ofthe primer, written in the 5′-3′ direction, were respectivelycomplementary in part to positions 1562–1537 of the chicken MMP-2sequence in the figure. A second downstream primer, designed to encode achicken MMP-2 protein termination site at position 552, to encode acysteine residue at position 551, and to contain an internal EcoRIendonuclease restriction site for insertion into a GST vector, had thenucleotide sequence (5′TCTGAATTCTGCCACAGTTGAAGG3′ (SEQ ID NO 36)). The5′ and 3′ ends of the primer, written in the 5′-3′ direction, wererespectively complementary in part to positions 1666–1643 of the chickenMMP-2 sequence in the figure. A third downstream primer, designed toencode a chicken MMP-2 protein termination site at position 637 and tocontain an internal EcoRI endonuclease restriction site for insertioninto a GST vector, had the nucleotide sequence(5′ATTGAATTCTTCTACAGTTCA3′ (SEQ ID NO 37)). The 5′ and 3′ ends of theprimer, written in the 5′-3′ direction, were respectively complementaryin part to positions 1932–1912 of the chicken MMP-2 sequence in thefigure.

The regions of the chicken MMP-2 carboxy terminus bonded by the aboveupstream and downstream primers, used in particular combinations toproduce the fusion proteins containing at least one engineered cysteineresidue as described above, were separately amplified for 30 cycles withan annealing temperature of 55C according to the manufacturer'sinstructions for the Expand High Fidelity PCR System (BoehringerMannheim). The resulting amplification products were separatelypurified, digested with BamHI and or EcoRI restriction enzymes asnecessary, and repurified before ligation into the appropriate GSTfusion protein vector, either pGEX-3X or pGEX-1λT, as indicated above bythe reading frame of the upstream oligonucleotide primer. For ligatingthe amplified MMP-2 products, the vectors were similarly digested aswell as dephosphorylated prior to the ligation reaction. Competent E.coli strain BL21 cells were then separately transformed with theresultant MMP-2-containing vector constructs by heat shock. Resultingcolonies were then screened for incorporation of the appropriate fusionprotein-encoding plasmid by PCR and production of the appropriate sizedGST-fusion protein prior to dideoxy sequencing of positive clones toverify the integrity of the introduced coding sequence. Purification ofrecombinant GST fusion proteins were the performed using IPTG-inducedlog-phase cultures essentially as described above for producing theother GST-MMP-2 fusion proteins.

In addition to the chicken MMP-2 GST-fusion proteins described above,two human MMP-2 GST fusion proteins were produced for expressing aminoacid regions 203–631 and 439–631 of the mature human MMP-2 proenzymepolypeptide. The indicated regions correspond respectively to chickenMMP-2 regions 203–637 and 445–637. Human MMP-2-GST fusion proteins wereproduced by PCR as described above for the chicken MMP-2-GST fusionproteins utilizing a cDNA template that encoded the entire human MMP-2open reading frame provided by Dr. W. G. Stetler-Stevenson at theNational Cancer Institute, Bethesda, Md. Upstream 5′ primer sequenceswere designed based upon the previously published sequence of humanMMP-2 (Collier et al., J. Biol. Chem., 263:6579–6587 (1988) and toencode an introduced internal EcoRI restriction site to allow forinsertion of the amplified products into the appropriate expressionvector.

One upstream primer, designed to encode a human MMP-2 protein start siteat position 203 after an engineered internal EcoRI endonucleaserestriction site for insertion into the pGEX-1λT GST vector, had thenucleotide sequence (5′GATGAATTCTACTGCAAGTT3′ (SEQ ID NO 38)). The 5′and 3′ ends of the primer respectively corresponded to positions 685–704of the human MMP-2 open reading frame sequence. Another upstream primer,designed to encode a human MMP-2 protein start site at position 439after an engineered internal EcoRI restriction site for insertion intothe pGEX-1λT GST vector, had the nucleotide sequence(5′CACTGAATTCATCTGCAAACA3′ (SEQ ID NO 39)). The 5′ and 3′ ends of theprimer respectively corresponded to positions 1392 and 1412 of the humanMMP-2 open reading frame sequence.

Each of the above primers were used separately with a downstream primer,having 5′ and 3′ ends respectively complementary to bases 1998 and 1978of the human MMP-2 sequence that ends distal to the MMP-2 open readingframe and directs protein termination after amino acid residue 631. Theamplified products produced expressed fusion proteins containing humanMMP-2 amino acid residues 203–631 (SEQ ID NO 40) and 439–631 (SEQ ID NO12).

The resulting PCR products were purified, digested with EcoRI andrepurified for ligation into a pGEX-1λT plasmid that was similarlydigested and dephosphorylated prior to the ligation reaction. Cells weretransformed as described above.

Other human MMP-2 fusion proteins containing amino acid residues 410–631(SEQ ID NO 11), 439–512 (SEQ ID NO 13), 439–546 (SEQ ID NO 14), 510–631(SEQ ID NO 15) and 543–631 (SEQ ID NO 16) are also prepared as describedabove for use in the methods of this invention.

B. Ligand-Receptor Binding Assay

The α_(v)β₅-immunoreactive antibodies and synthetic peptides preparedrespectively in Examples 1 and 3 are screened by measuring their abilityto antagonize α_(v)β₅, α_(v)β₃, and α_(IIb)β₃ receptor binding activityin purified ligand-receptor binding assays. The method for these bindingstudies has been described by Barbas et al., Proc. Natl. Acad. Sci.,USA, 90:10003–10007 (1993), Smith et al., J. Biol. Chem.,265:11008–11013 (1990) and Pfaff et al., J. Biol. Chem., 269:20233–20238(1994), the disclosures of which are hereby incorporated by reference.

A method of identifying antagonists in a ligand-receptor binding assayis described in which the receptor is immobilized to a solid support andthe ligand and antagonist are soluble. A ligand-receptor binding assayis also described in which the ligand is immobilized to a solid supportand the receptor and antagonists are soluble.

Briefly, selected purified integrins are separately immobilized inTitertek microtiter wells at a coating concentration of 50 nanograms(ng) per well. The purification of the receptors used in theligand-receptor binding assays are well known in the art and are readilyobtainable with methods familiar to one of ordinary skill in the art.After incubation for 18 hours at 4C, nonspecific binding sites on theplate are blocked with 10 milligrams/milliliter (mg/ml) of bovine serumalbumin (BSA) in Tris-buffered saline. For inhibition studies, variousconcentrations of selected antibodies or peptides are tested for theability to block the binding of ¹²⁵I-vitronectin or other labeledligands to the integrin receptors, α_(v)β₅, α_(v)β₃, α_(v)β₁ andα_(IIb)β₃.

Although these ligands exhibit optimal binding for a particularintegrin, vitronectin for α_(v)β₅ and α_(v)β₃ and fibrinogen forα_(IIb)β₃, inhibition of binding studies using either antibodies orpeptides to block the binding of vitronectin to either receptor allowsfor the accurate determination of the amount in micromoles (μM) ofpeptide necessary to half-maximally inhibit the binding of receptor toligand. Radiolabeled ligands are used at concentrations of 1 nM andbinding is challenged separately with unlabeled synthetic peptides.Following a three hour incubation, free ligand is removed by washing andbound ligand is detected by gamma counting.

Thus, the ligand-receptor assay described herein is used to screen forboth circular or linearized synthetic peptides along with monoclonalantibodies and organic molecules that exhibit selective specificity fora particular integrin receptor, specifically α_(v)β₅, as used asvitronectin receptor (α_(v)β₅) antagonists in practicing this invention.

8. In Vivo Regression of Tumor Tissue Growth With α_(v)β₅ Antagonists asMeasured by Chimeric Mouse:Human Assay

An in vivo chimeric mouse:human model was generated by replacing aportion of skin from a SCID mouse with human neonatal foreskin. The invivo chimeric mouse:human model was prepared essentially as described inYan, et al., J. Clin. Invest., 91:986–996 (1993). Briefly, a 2 cm²square area of skin was surgically removed from a SCID mouse (6–8 weeksof age) and replaced with a human foreskin. The mouse was anesthetizedand the hair removed from a 5 cm² area on each side of the lateralabdominal region by shaving. Two circular graft beds of 2 cm² wereprepared by removing the full thickness of skin down to the fascia. Fullthickness human skin grafts of the same size derived from human neonatalforeskin were placed onto the wound beds and sutured into place. Thegraft was covered with a Band-Aid which was sutured to the skin.Micropore cloth tape was also applied to cover the wound.

After the skin graft was established, the human foreskin was inoculatedwith melanoma cells. The M21L human melanoma cell line was used to formthe solid human tumors on the human skin grafts on the SCID mice. Asingle cell suspension of 2×10⁶ M21L was injected intradermally into thehuman skin graft. The mice were then observed for 2 to 4 weeks to allowgrowth of measurable human tumors.

After a measurable tumor was established, either 250 μg of the peptide(in a volume of 100 μl) having SEQ ID NO 9 (cyclic RGD-containingpeptide Arg-Gly-Asp-D-Phe-Asn-NMeVAL) or a control peptide, cycloArg-βAla-Asp-D-Phe-Val, were injected intraperitoneally into the mouse 3times per week over 3 weeks. At the end of this time, the tumor wasexcised and analyzed by weight and histology.

The results are shown in FIG. 9 where the tumor volume in mm³ is plottedon the Y-axis against the peptide treatments on the X-axis. The testpeptide having SEQ ID NO 9, labeled in the figure as peptide 189,significantly reduced the tumor volume to approximately 25 mm³ comparedto control peptide (labeled as peptide 601) where the tumor volume wasgreater than 300 mm³.

Thus, the blocking of the α_(v)β₅ receptor by the intravenousapplication of α_(v)β₅ antagonist peptide 189 resulted in a regressionof a melanoma tumor in this model system in the same manner as the CAMand rabbit eye model systems as described previously.

In other experiments with M21-L melanoma tumor cells in the mouse:humanchimeric assay system, the response with mAB LM609 was compared with theresponse obtained with the synthetic peptide 85189 (SEQ ID NO 9) ascompared to control synthetic peptide 69601 (SEQ ID NO 5). The assayswere performed as described above. The results, shown in FIG. 21,demonstrate that the synthetic peptide 85189 reduced tumor volume tobelow 25 mm³ as compared to control peptide where the tumor volume wasapproximately 360 mm³. The mAB LM609 also significantly reduced tumorvolume to approximately 60 mm³.

Thus, blocking of the α_(v)β₃ receptor by the intravenous application ofα_(v)β₃-specific LM609 antibody and peptides resulted in a regression ofa carcinoma in this model system as compared to the other model systemsdescribed in this invention.

In additional assays with the SCID mice model having measurable M21-Ltumors, in a preliminary analysis, a dose response curve was performedfor peptides 69601 (control) and 85189 (test) injected over aconcentration range of 10 to 250 μg/ml. The mean volume and weight ofresected tumors following treatment were determined with the resultsrespectively shown in FIGS. 22A and 22B. Peptide 85189 was effective atinhibiting M21-L tumor growth over the concentration range testedcompared to treatment with control peptide with the most effectivedosage being 250 μg/ml.

For analyzing peptide 85189 treatment effectiveness over a time course,two treatment regimens were evaluated in the same SCID tumor model. Inone assay, treatment with either peptide 85189 or 69601 was initiated onday 6, with day 0 being the day of M21-L tumor injection of 3×10⁶ cellssubcutaneously into mouse skin, with intraperitoneal injections of 250μg/ml peptide 85189 or control 69601 every other day until day 29. Theother assay was identically performed with the exception that treatmentwas initiated on day 20. At the end of the assays, the tumors wereresected and the mean tumor volume in mm³ was determined. The data wasplotted as this value +/− the standard error of the mean.

The results of these assays, respectively shown in FIGS. 23A and 23B,indicate that peptide 85189 but not 69601 inhibited tumor growth atvarious days after treatment was initiated, depending on the particulartreatment regimen. Thus, peptide 85189 is an effective α_(v)β₅antagonist of both angiogenesis and thus tumor growth.

The SCID/human chimeric model above is also used for assessing theeffectiveness of other α_(v)β₅ antagonists of this invention, namelyantibodies. MMP-2 preparations, previously prepared, and organicmolecules, the latter of which are prepared as described in Example 10.

9. Preparation of a Murine Mouse Model for α_(v)β₅-Mediated RetinalAngiogenesis and Inhibition Thereof with α_(v)β₅ Antagonists

Based on the observation in Example 2C of α_(v)β₃ and α_(v)β₅ expressionin retinal neovascular tissue, a novel mouse model was used to study theeffects of systemically administered cyclic peptide antagonists of bothintegrins on retinal angiogenesis. Newborn mice develop retinal vesselsduring the first two weeks postnatally during which time the superficialretinal vasculature forms a rich, highly branched network of vesselsthat originate at the optic nerve head and radiate peripherally to coverthe retinal surface in a manner similar to that observed in othermammals and humans (Jiang et al., Glia. 15:1–10 (1995).

For the model, newborn mice were injected subcutaneously twice daily forfour days starting from day 0 with the cyclic peptide RGDfV (SEQ ID NO4) (also referred to as peptide 203) or the control peptide RADfV (SEQID NO 5). On postnatal day five, globes were removed and fixed in 4.0%paraformaldehyde (PFA) at room temperature.

To quantitate mouse retinal angiogenesis, the distance from the opticnerve head to the most distal point of a single vessel selected in eachof six equal sectors around a twelve hour clock was measured. The meandistance was calculated and averaged with similar data obtained from anentire litter. To measure the total volume of retinal blood vessels, theentire specimen was scanned in 2.0 μm optical sections and storeddigitally. The “seed” function in Bio-Rad's Lasersharp software was thenused to threshold and count cubic pixels in each section. A macro waswritten to sum the volume of all sections and determine the value forall vascular structures.

With the direct measurement of vessel growth in two dimensions fromphotographs, systemically, administered peptide antagonist 203 inhibitedretinal vasculogenesis, relative to control peptide, by 44% (N=9,p<0.0000001, paired t-test). No statistical difference was seen betweenuntreated newborn mice and five-day old mice receiving peptide 203, thusthe peptide effectively inhibits vasculogenesis. In addition, nostatistical difference was seen between untreated five-day old mice andthe same aged mice receiving control peptide. Thus, inhibition ofretinal vasculogenesis in RGDfV-treated newborn mice when compared tountreated counterparts is effectively 100%.

Using a more quantitative analysis taking the three dimensional natureof vessel growth, a 78% reduction in the retinal vascular volume in thepeptide 203-treated animals compared to the controls was seen. The meanvolume of vessels on postnatal day five in 203-treated animals was3.6×10⁶ μm³ and in control-treated animal was 15.7×10⁶ μm³. The volumeoccupied by retinal blood vessels in untreated newborn mice wasindistinguishable from the five-day old 203-treated animals.

The results obtained above showed that the antagonists specificallyblocked new blood vessel formation with no effect on establishedvessels. The results indicate that the pathology of retinal neovasculardisease is distinct from that seen with subretinal neovascular diseaseand that antagonists of α_(v)β₅ are effective for treating patients withblinding eye disease associated with angiogenesis.

Similar assays are performed with the MMP-2 and organic mimetic α_(v)β₅antagonists prepared as respectively described in Example 7 and Example10.

10. Preparation of Organic Molecule α_(v)β₅ Antagonists

The synthesis of organic α_(v)β₅ antagonist Compounds 7, 9, 10, 12, 14,15, 16, 17 and 18 is described below and is also shown in the notedfigures. The resultant organic molecules, referred to as organicmimetics of this invention, are then used in the methods for inhibitingα_(v)β₅-mediated angiogenesis.

For each of the syntheses described below, optical rotations weremeasured on Perkin-Elmer 241 spectrophotometer UV and visible spectrawere recorded on a Beckmann DU-70 spectrometer. ¹H and ¹³C NMR spectrawere recorded at 400 and 500 MHz on Bruker AMX-400 and AMX-500spectrometer. High-resolution mass spectra (HRMS) were recorded on a VGZAB-ZSE mass spectrometer under fast atom bombardment (FAB) conditions.Column chromatography was carried out with silica gel of 70–230 mesh.Preparative TLC was carried out on Merck Art. 5744 (0.5 mm). Meltingpoints were taken on a Thomas Hoover apparatus.

A. Compound 1; t-Boc-L-tyrosine Benzyl Ester as Illustrated in FIG. 10

To a solution of N-(tert-butoxycarbonyl)-L-tyrosine(t-Boc-L-tyrosine)(1.0 equivalents; Aldrich) in 0.10 M (M) methylene chloride was addeddicyclohexylcarbodiimide (DCC) (1.5 equivalents) at 25° C. and allowedto stir for 1 hour. Next, 1.5 equivalents benzyl alcohol was added andthe mixture was stirred for an additional 12 hours at 25° C. Thereaction mixture was then diluted with ethyl acetate (0.10 M) and washedtwice (2×) with water, once (1×) with brine and dried over magnesiumsulfate. The solvent was then removed in vacuo and the crude product wasthen purified by silica gel column chromatography. Compound 1,t-Boc-L-tyrosine benzyl ester can also be commercially purchased fromSigma.

B. Compound 2;(S)-3-(4-(4-Bromobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 10 Step i

A mixture of t-Boc-L-tyrosine benzyl ester (2 grams, 5.38 mmol;synthesized as described above), 1,4-dibromobutane (1.9 ml, 16.2 mmol;Aldrich), potassium carbonate (5 g) and 18-crown-6 (0.1 g; Aldrich), washeated at 80° C. for 12 hours. After cooling, the precipitate wasfiltered off and the reaction mixture was evaporated to dryness invacuo. The crude product was then purified by crystallization using 100%hexane to yield 2.5 g (92%) of Compound 2.

C. Compound 3;(S)-3-(4-(4-Azidobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 10 Step ii

Compound 2 (2.5 g, 4.9 mmol) was stirred with sodium azide (1.6 g, 25mmol) in dimethylformamide (DMF) (20 ml) at 25° C. for 12 hours. Thesolvent was then evaporated and the residue was treated with water(approx 10 ml) and extracted twice with ethyl acetate. The organiclayers were combined, dried via magnesium sulfate and evaporated toyield 2.0 grams (90%) of Compound 3 as a colorless syrup (FAB-MS: 469(M+H⁺).

D. Compound 4; (S)-3-(4-(4-Azidobutyloxy)phenyl-2-amino-propionic AcidBenzyl Ester as Illustrated in FIG. 10 Step iii

Compound 3 (2.0 g (4.4 mmol)) was dissolved in trifluoroacetic acid(TFA; 2 ml) and stirred for 3 hours at room temperature. Evaporation invacuo yielded 1.6 grams (quantitative) of Compound 4 as a colorlesssyrup that was used without further purification for the next step.FAB-MS: 369 (M⁺H⁺).

E. Compound 5;(S)-3-(4-(4-Azidobutyloxy)phenyl-2-butylsulfonamido-propionic AcidBenzyl Ester as Illustrated in FIG. 10 Step iv

A mixture of Compound 4 (1.6 g; 4.3 mmol), butane sulfonic acid chloride(0.84 ml; 6.6 mmol) and triethyl amine (1.5 equivalents) were stirred inmethylene chloride (20 ml) for 12 hours at room temperature. Thereaction mixture was then evaporated and the residue was dissolved inethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yield1.4 grams (67%) of Compound 5 as an amorphous solid.

F. Compound 6;(S)-3-(4-(4-Aminobutyloxy)phenyl-2-butylsulfonamido-propionic Acid asIllustrated in FIG. 10 Step v

Compound 5 (1.3 g (2.6 mmol) was dissolved in 20 ml of ethylacetate/methanol/water 5/3/1 and 0.2 ml trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25°C. in the presence of 100 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldCompound 6 as an oily residue. After lyophilization from water 1.0 gram(quantitative) of Compound 6 was obtained as a white powder. FAB-MS: 373(M⁺H⁺).

G. Compound 7;(S)-3-(4-(4-Guanidinobutyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 10 Step vi

Compound 6 (200 mg; 0.5 mmol), 3,5-dimethylpyrazol-1-carboxamidinenitrate (DPFN) (170 mg; 0.8 mmol; Aldrich Chemical Company) andtriethylamine (0.15 ml, 1.0 mmol) in dimethylformamide (DMF; 5 ml) wereheated at 60° C. for 12 hours. After cooling, the solvent was evaporatedin vacuo, and the residue was purified by HPLC (Lichrocart RP-18,gradient acetonitrile/water+0.3% TFA 99:1 to 1:99) to yield 50 mg (25%)of Compound 7 as a white, amorphous powder, after lyophilization.FAB-MS: 415 (M⁺H⁺), m.p.: 70° C.

H. Compound 8;(S)-3-(4-(4-Aminobutyloxy)phenyl-2-N-tert,butyloxycarbonyl-propionicAcid as Illustrated in FIG. 11 Step iii

Compound 3 (0.5 g (1.07 mmol) was dissolved in 10 ml of ethylacetate/methanol/water 5/3/1 and 0.1 ml trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25°C. in the presence of 30 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldCompound 8 as an oily residue. After lyophilization from water 370milligram (quantitative) of Compound 8 was obtained as a white powder.FAB-MS: 353 (M⁺H⁺).

I. Compound 9;(S)-3-(4-(4-Guanidinobutyloxy)phenyl-2-N-tert,butyloxycarbonyl-propionicAcid as Illustrated in FIG. 11 Step iv

Compound 8 (200 mg; 0.5 mmol), 3,5-dimethylpyrazol-1-carboxamidinenitrate (DPFN) (170 mg; 0.8 mmol; Aldrich Chemical Company) andtriethylamine (0.15 ml, 1.0 mmol) in dimethylformamide (DMF; 5 ml) wereheated at 60° C. for 12 hours. After cooling, the solvent was evaporatedin vacuo, and the residue was purified by HPLC (Lichrocart RP-18,gradient acetonitrile/water+0.3% TFA 99:1 to 1:99) to yield 160 mg (90%)of Compound 9 as a white, amorphous powder, after lyophilization.FAB-MS: 395 (M⁺H⁺).

J. Compound 10;(R)-3-(4-(4-Guanidinobutyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 12 Steps i–vi

The identical reaction sequence to synthesize Compound 7 was used toprepare the D-tyrosine analog 10 of which 205 mg were obtained as awhite amorphous material FAB-MS: 415 (M⁺H⁺) as follows usingintermediate Compounds 100–600 to form Compound 10:

1) Compound 100; t-Boc-D-tyrosine Benzyl Ester as Illustrated in FIG. 12

To a solution of N-(tert-butoxycarbonyl)D-tyrosine(t-Boc-L-tyrosine)(1.0 equivalents; Aldrich) in 0.10 M methylene chloride was addeddicyclohexylcarbodiimide (DCC) (1.5 equivalents) at 25° C. and allowedto stir for 1 hour. Next, 1.5 equivalents benzyl alcohol was added andthe mixture was stirred for an additional 12 hours at 25° C. Thereaction mixture was then diluted with ethyl acetate (0.10 M) and washed2× with water, 1× with brine and dried over magnesium sulfate. Thesolvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography.

2) Compound 200;(R)-3-(4-(4-Bromobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 12 Step i

A mixture of t-Boc-D-tyrosine benzyl ester (2 grams, 5.38 mmol;synthesized as described above), 1,4-dibromobutane (1.9 ml, 16.2 mmol;Aldrich), potassium carbonate (5 g) and 18-crown-6 (0.1 g; Aldrich), washeated at 80° C. for 12 hours. After cooling, the precipitate wasfiltered off and the reaction mixture was evaporated to dryness invacuo. The crude product was then purified by crystallization using 100%hexane to yield 2.5 g (92%) of Compound 200.

3) Compound 300;(R)-3-(4-(4-Azidobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 12 Step ii

Compound 200 (2.5 g, 4.9 mmol) was stirred with sodium azide (1.6 g, 25mmol) in dimethylformamide (DMF) (20 ml) at 25° C. for 12 hours. Thesolvent was then evaporated and the residue was treated with water(approx 10 ml) and extracted twice with ethyl acetate. The organiclayers were combined, dried via magnesium sulfate and evaporated toyield 2.0 grams (90%) of Compound 300 as a colorless syrup (FAB-MS: 469(M+H⁺).

4) Compound 400; (R)-3-(4-(4-Azidobutyloxy)phenyl-2-amino-propionic AcidBenzyl Ester as Illustrated in FIG. 12 Step iii

Compound 300 (2.0 g (4.4 mmol)) was dissolved in trifluoroacetic acid(TFA; 2 ml) and stirred for 3 hours at room temperature. Evaporation invacuo yielded 1.6 grams (quantitative) of Compound 400 as a colorlesssyrup that was used without further purification for the next step.FAB-MS: 369 (M⁺H⁺).

5) Compound 500;(R)-3-(4-(4-Azidobutyloxy)phenyl-2-butylsulfonamido-propionic AcidBenzyl Ester as Illustrated in FIG. 12 Step iv

A mixture of Compound 400 (1.6 g; 4.3 mmol), butane sulfonic acidchloride (0.84 ml; 6.6 mmol) and triethyl amine (1.5 equivalents) werestirred in methylene chloride (20 ml) for 12 hours at room temperature.The reaction mixture was then evaporated and the residue was dissolvedin ethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yield1.4 grams (67%) of Compound 500 as an amorphous solid.

6) Compound 600;(R)-3-(4-(4-Aminobutyloxy)phenyl-2-butylsulfonamido-propionic Acid asIllustrated in FIG. 12 Step v

Compound 500 (1.3 g (2.6 mmol) was dissolved in 20 ml of ethylacetate/methanol/water 5/3/1 and 0.2 ml trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25°C. in the presence of 100 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldCompound 600 as an oily residue. After lyophilization from water 1.0gram (quantitative) of Compound 600 was obtained as a white powder.FAB-MS: 373 (M⁺H⁺).

7) Compound 10;(R)-3-(4-(4-Guanidinobutyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 12 Step vi

Compound 600 (200 mg; 0.5 mmol), 3,5-dimethylpyrazol-1-carboxamidinenitrate (DPFN) (170 mg; 0.8 mmol; Aldrich Chemical Company) andtriethylamine (0.15 ml, 1.0 mmol) in dimethylformamide (DMF; 5 ml) wereheated at 60° C. for 12 hours. After cooling, the solvent was evaporatedin vacuo, and the residue was purified by HPLC (Lichrocart RP-18,gradient acetonitrile/water+0.3% TFA 99:1 to 1:99) to yield 50 mg (25%)of Compound 10 as a white, amorphous powder, after lyophilization.FAB-MS: 415 (M⁺H⁺), m.p.: 70° C.

K. Compound 11;(S)-3-(4-(4-Azidobutyloxy)phenyl-2-(10-camphorsulfonamido)-propionicAcid Benzyl Ester as Illustrated in FIG. 4

A mixture of Compound 4 (1.0 g; 2.7 mmol), 10-camphorsulfonic acidchloride (6.6 mmol; Aldrich Chemical Company) and triethyl amine (1.5equivalents) were stirred in methylene chloride (20 mL) for 12 hours atroom temperature. The reaction mixture was then evaporated and theresidue was dissolved in ethylacetate, washed with dilute HCl, aqueoussodium bicarbonate and water. After evaporation to dryness the crudeproduct was purified by flash chromatography (silica gel,toluene/ethylacetate 15:1) to yield 1.4 grams (67%) of Compound 11 as anamorphous solid.

L. Compound 12;(S)-3-(4-(4-Guanidinobutyloxy)phenyl-2-(10-camphorsulfonamido)-propionicAcid as Illustrated in FIG. 4 Steps i–ii

Compound 12 is obtained after hydrogenation and guanylation of compound11 according to the following conditions:

Step i: Compound 11 (1.3 g (2.6 mmol) was dissolved in 20 mL of ethylacetate/methanol/water 5/3/1 and 0.2 mL trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25°C. in the presence of 100 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldthe intermediate amine as an oily residue. After lyophilization fromwater 1.0 gram (quantitative) of the intermediate amine was obtained asa white powder, which was carried on as follows:

Step ii: The above formed intermediate amine compound (200 mg; 0.5mmol), 3,5-dimethylpyrazol-1-carboxamidine nitrate (DPFN) (170 mg; 0.8mmol; Aldrich Chemical Company) and triethylamine (0.15 mL, 1.0 mmol) indimethylformamide (DMF; 5 mL) were heated at 60° C. for 12 hours. Aftercooling, the solvent was evaporated in vacuo, and the residue waspurified by HPLC (Lichrocart RP-18, gradient acetonitrile/water+0.3% TFA99:1 to 1:99) to yield 50 mg (25%) of compound 12 as a white, amorphouspowder, after lyophilization. FAB-MS: 509.6 (M+H+).

M. Compound 13;(S)-3-(4-(5-Bromopentyloxy)phenyl-2-N-tert,butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 13

A mixture of t-Boc-L-tyrosine benzyl ester (4.5 grams, 12.1 mmol;Compound 1 synthesized as described above), 1,5-dibromopentane (5 ml,36.7 mmol; Aldrich), potassium carbonate (10 g) and 18-crown-6 (0.25 g;Aldrich), was heated at 80° C. for 12 hours. After cooling, theprecipitate was filtered off and the reaction mixture was evaporated todryness in vacuo. The crude product was then purified by crystallizationusing 100% hexane to yield 5.35 g (85%) of Compound 13.

N. Compound 14;(S)-3-(4-(5-Guanidinopentyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 13 Steps i–v

The 5 step reaction sequence of bromine-azide-exchange, Boc-cleavage,sulfonylation with butane sulfonic acid chloride, hydrogenation andguanylation with DPFN was carried out identically to the aboveprocedures using intermediate Compounds 1–6 to form Compound 7 or theprocedures using Compounds 100–600 to form Compound 10, as discussedabove. Compound 14 was obtained as a white powder FAB-MS: 429 (M⁺H⁺).

O. Compound 15;3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-aminoethyl)phenoxy)methyl-2-oxazolidinone,Dihydrochloride as Shown in FIG. 14

1) Synthesis of Starting Material2-N-Boc-amino-3-(4-hydroxyphenyl)propionate for Compound 15

The starting material 2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate wasobtained via esterification of (D or L),N-(tert-butoxycarbonyl)-L(D)-tyrosine (t-Boc-L(D)-tyrosine) (1.0equivalents; Sigma) in 0.10 M methanol and dilute 1% HCl. The reactionmixture was stirred at 25° C. for 12 hours and then neutralized viapotassium carbonate and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× brine and dried over magnesium sulfate. Thesolvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate.

2) Synthesis of Starting Material3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 15; 3-Step Procedure as Follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25° C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25° C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110° C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25° C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25° C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0° C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of Intermediates 2-N-BOC-amino-3-(4-hydroxyphenyl)propionatewith3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toForm Protected Form of Compound 15,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2N-BOC-aminoethyl)phenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams 2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate (asdescribed above), 20 ml dimethylformamide (DMF) and NaH (1.0equivalent), was stirred for 30 minutes at room temperature. Afterstirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 15,3-(4-BOC-amidinophenyl)-5-(4-methoxy-carbonyl-2N-BOC-aminoethyl)phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of Protected Form of Compound 15 to Form Compound 15:3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-amino-ethyl)phenoxy)methyl-2-oxazolidinone,Dihydrochloride, FIG. 14

Treatment of the protected form of Compound 15,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2N-BOC-aminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0° C. to 25° C. for 3 hours.The reaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 15:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-amino-ethyl)phenoxy)methyl-2-oxazolidinone,dihydrochloride; m.p. 165° C. (d).

P. Compound 16;3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-N-butylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinoneas Shown in FIG. 14

1) Synthesis of Starting Material2-N-butylsulfonylamino-3-(4-hydroxyphenyl)propionate for Compound 16

The starting material2-N-butylsulfonylamino-3-(4-hydroxy-phenyl)propionate was obtained viaesterification of ((D or L) tyrosine) (1.0 equivalents; Sigma) in 0.10 Mmethanol and dilute 1% HCl. The reaction mixture was stirred at 25° C.for 12 hours and then neutralized via potassium carbonate and thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then carried on as follows:

A mixture of the above compound (4.3 mmol), butane sulfonic acidchloride (6.6 mmol) and triethyl amine (1.5 equivalents) were stirred inmethylene chloride (20 ml) for 12 hours at room temperature. Thereaction mixture was then evaporated and the residue was dissolved inethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yieldthe title compound.

2) Synthesis of Starting Material3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 16; 3-Step Procedure as Follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25° C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25° C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110° C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25° C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25° C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0° C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of Intermediates2-N-butylsulfonylamino-3-(4-hydroxyphenyl)propionate with3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toForm Protected Form of Compound 16,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-butylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams2-N-butylsulfonylamino-3-(4-hydroxy-phenyl)propionate (as describedabove), 20 ml dimethylformamide (DMF) and NaH (1.0 equivalent), wasstirred for 30 minutes at room temperature. After stirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 16,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-butylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of Protected Form of Compound 16 to Form Compound 16;3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-N-butylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone,FIG. 14

Treatment of the protected form of Compound 16,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-butylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0° C. to 25° C. for 3 hours.The reaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 16:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-butylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone;m.p. 236–237° C.

Q. Compound 17;3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-N-propylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinoneas Shown in FIG. 14

1) Synthesis of Starting Material2-N-propyl-sulfonylamino-3-(4-hydroxyphenyl)propionate for Compound 17;

The starting material2-N-propyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate was obtained viaesterification of ((D or L) tyrosine) (1.0 equivalents; Sigma) in 0.10 Mmethanol and dilute 1% HCl. The reaction mixture was stirred at 25° C.for 12 hours and then neutralized via potassium carbonate and thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then carried on as follows:

A mixture of the above compound (4.3 mmol), propyl sulfonic acidchloride (6.6 mmol; Aldrich) and triethyl amine (1.5 equivalents) werestirred in methylene chloride (20 ml) for 12 hours at room temperature.The reaction mixture was then evaporated and the residue was dissolvedin ethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yieldthe title compound.

2) Synthesis of Starting Material3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 17; 3-Step Procedure as Follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25° C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25° C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110° C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25° C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25° C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0° C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of Intermediates2-N-propyl-sulfonylamino-3-(4-hydroxyphenyl)propionate with3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toForm Protected Form of Compound 17,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-propyl-sulfonylaminoethyl)-phenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams2-N-propyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate (as describedabove), 20 ml dimethylformamide (DMF) and NaH (1.0 equivalent), wasstirred for 30 minutes at room temperature. After stirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 17,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-propyl-sulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of Protected Form of Compound 17 to Form Compound 17;3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-N-propylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone,FIG. 14

Treatment of the protected form of Compound 17,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-propylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0° C. to 25° C. for 3 hours.The reaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 17:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-propylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone;m.p. 200° C. (d).

R. Compound 18;3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-N-ethylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinoneas Shown in FIG. 14

1) Synthesis of Starting Material2-N-ethyl-sulfonylamino-3-(4-hydroxyphenyl)propionate for Compound 18;

The starting material2-N-ethyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate was obtained viaesterification of ((D or L) tyrosine) (1.0 equivalents; Sigma) in 0.10 Mmethanol and dilute 1% HCl. The reaction mixture was stirred at 25° C.for 12 hours and then neutralized via potassium carbonate and thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then carried on as follows:

A mixture of the above compound (4.3 mmol), ethyl sulfonic acid chloride(6.6 mmol; Aldrich) and triethyl amine (1.5 equivalents) were stirred inmethylene chloride (20 ml) for 12 hours at room temperature. Thereaction mixture was then evaporated and the residue was dissolved inethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yieldthe title compound.

2) Synthesis of Starting Material3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 18; 3-Step Procedure as Follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25° C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25° C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110° C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25° C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25° C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0° C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of Intermediates2-N-ethyl-sulfonylamino-3-(4-hydroxyphenyl)propionate with3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toForm Protected Form of Compound 18,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-ethyl-sulfonylaminoethyl)-phenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams2-N-ethyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate (as describedabove), 20 ml dimethylformamide (DMF) and NaH (1.0 equivalent), wasstirred for 30 minutes at room temperature. After stirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 18,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-ethyl-sulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of Protected Form of Compound 18 to Form Compound 18;3-(4-Amidinophenyl)-5-(4-(2-carboxy-2-N-ethylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone,FIG. 14

Treatment of the protected form of Compound 18,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-ethylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0° C. to 25° C. for 3 hours.The reaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 18:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-ethylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone;m.p. 212° C. (d).

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. A method for inhibiting α_(v)β₅ mediated angiogenesis in a tissuecomprising administering to said tissue a composition comprising anangiogenesis-inhibiting amount of an α_(v)β₅ antagonist, wherein saidantagonist is a matrix metalloproteinase polypeptide consisting of: SEQID NO 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or
 22. 2. The method ofclaim 1 wherein said tissue is human tissue.
 3. The method of claim 2wherein said tissue is inflamed and said angiogenesis is inflamed tissueangiogenesis.
 4. The method of claim 1 wherein said tissue is arthritic.5. The method of claim 4 wherein said arthritic tissue is present in amammal with rheumatoid arthritis.
 6. The method of claim 1 wherein saidtissue is the retinal tissue and said angiogenesis is retinalangiogenesis.
 7. The method of claim 6 wherein said retinal tissue is ina patient with diabetic retinopathy or macular degeneration.
 8. Themethod of claim 1 wherein said tissue is a solid tumor or a solid tumormetastasis and said angiogenesis is tumor angiogenesis.
 9. The method ofclaim 8 wherein said tissue is a carcinoma.
 10. The method of claim 8wherein said solid tumor is a tumor of lung, pancreas, breast, colon,larynx or ovary.
 11. The method of claim 8 wherein said administering isconducted in conjunction with chemotherapy.
 12. The method of claim 1wherein said administering comprises intravenous, transdermal,intrasynovial, intramuscular, or oral administration.
 13. The method ofclaim 1 wherein said angiogenesis-inhibiting amount is from about 0.1mg/kg to about 300 mg/kg.
 14. The method of claim 1 wherein saidtherapeutically effective amount is from about 0.1 mg/kg to about 300mg/kg.
 15. The method of claim 1 wherein said administering comprises asingle dose intravenously.
 16. The method of claim 1 wherein saidadministering comprises one or more dose administrations daily for oneor more days.
 17. The method of claim 1 wherein said angiogenesis ispresent in a patient having an eye disease selected from the group ofeye diseases consisting of diabetic retinopathy, age-related maculardegeneration, presumed ocular histoplasmosis, retinopathy of prematurityand neovascular glaucoma.
 18. The method of claim 1 wherein saidangiogenesis is present in a patient having a corneal neovasculardisorder selected from the group of disorders consisting of cornealtransplantation, herpetic keratitis, luetic keratitis, pterygium andneovascular pannus associated with contact lens use.
 19. The method ofclaim 1 wherein said angiogenesis is induced by a cytokine.
 20. Themethod of claim 19 wherein said cytokine is selected from the groupconsisting of vascular endothelial growth factor, transforming growthfactor-α and epidermal growth factor.
 21. The method of claim 19 whereinsaid cytokine is vascular endothelial growth factor and saidangiogenesis is selected from the group consisting of retinalangiogenesis, corneal angiogenesis, tumor angiogenesis and inflamedtissue angiogenesis.
 22. A method for inhibiting α_(v)β₅ mediatedangiogenesis in a tissue comprising administering to said tissue acomposition comprising an angiogenesis-inhibiting amount of an α_(v)β₅antagonist, wherein said antagonist is an organic compound selected fromthe group consisting of compounds represented by the followingstructures:


23. The method of claim 22 wherein said tissue is human tissue.
 24. Themethod of claim 23 wherein said tissue is inflamed and said angiogenesisis inflamed tissue angiogenesis.
 25. The method of claim 22 wherein saidtissue is arthritic.
 26. The method of claim 25 wherein said arthritictissue is present in a mammal with rheumatoid arthritis.
 27. The methodof claim 22 wherein said tissue is the retinal tissue and saidangiogenesis is retinal angiogenesis.
 28. The method of claim 27 whereinsaid retinal tissue is in a patient with diabetic retinopathy or maculardegeneration.
 29. The method of claim 22 wherein said tissue is a solidtumor or a solid tumor metastasis and said angiogenesis is tumorangiogenesis.
 30. The method of claim 29 wherein said tissue is acarcinoma.
 31. The method of claim 29 wherein said solid tumor is atumor of lung, pancreas, breast, colon, larynx or ovary.
 32. The methodof claim 29 wherein said administering is conducted in conjunction withchemotherapy.
 33. The method of claim 22 wherein said administeringcomprises intravenous, transdermal, intrasynovial, intramuscular, ororal administration.
 34. The method of claim 22 wherein saidangiogenesis-inhibiting amount is from about 0.1 mg/kg to about 300mg/kg.
 35. The method of claim 22 wherein said therapeutically effectiveamount is from about 0.1 mg/kg to about 300 mg/kg.
 36. The method ofclaim 22 wherein said administering comprises a single doseintravenously.
 37. The method of claim 22 wherein said administeringcomprises one or more dose administrations daily for one or more days.38. The method of claim 22 wherein said angiogenesis is present in apatient having an eye disease selected from the group of eye diseasesconsisting of diabetic retinopathy, age-related macular degeneration,presumed ocular histoplasmosis, retinopathy of prematurity andneovascular glaucoma.
 39. The method of claim 22 wherein saidangiogenesis is present in a patient having a corneal neovasculardisorder selected from the group of disorders consisting of cornealtransplantation, herpetic keratitis, luetic keratitis, pterygium andneovascular pannus associated with contact lens use.
 40. The method ofclaim 22 wherein said angiogenesis is induced by a cytokine.
 41. Themethod of claim 40 wherein said cytokine is selected from the groupconsisting of vascular endothelial growth factor, transforming growthfactor-α and epidermal growth factor.
 42. The method of claim 40 whereinsaid cytokine is vascular endothelial growth factor and saidangiogenesis is selected from the group consisting of retinalangiogenesis, corneal angiogenesis, tumor angiogenesis and inflamedtissue angiogenesis.